DNA sequences coding for a cinnamoyl CoA reductase and their uses in the field of regulating the lignin levels of plants

ABSTRACT

DNA sequences comprising, as coding region, all or part of the nucleotide sequence coding for an mRNA coding for a cinnamoyl CoA reductase (CCR), or all or part of the complementary nucleotide sequence thereof and coding for an anti-sense mRNA capable of hybridizing with the above-mentioned mRNA. The invention also concerns the use of said sequences for carrying out methods of regulation of plant lignin biosynthesis.

FIELD OF THE INVENTION

A subject of the present invention is the use of DNA sequences codingfor a cinnamoyl CoA reductase (CCR) in plants, or any fragment of thesesequences, or also any sequence derived from the latter, or theircomplementary sequences, within the scope of the implementation ofprocesses for the regulation of the lignin level in plants.

BACKGROUND OF THE INVENTION

Lignin is a complex heterogeneous aromatic polymer which waterproofs andreinforces the walls of certain plant cells.

Lignin is formed by the polymerization of free radicals derived frommonolignols such as paracoumarylic, coniferylic and sinapylic alcohols(Higuchi, 1985, in Biosynthesis and degradation of wood components (T.Higuchi, ed), Academic Press, Orlando, Fla. pp. 141-160).

Lignins have a large variation in their relative monolignol levels, as afunction of the species and the different tissues within the same plant.

This variation is probably due to and controlled by the differentactivities and specificities of substrates, of enzymes necessary for thebiosynthesis of lignin monomers (Higuchi, 1985, mentioned above).

Beyond its role in the structure and development of plants, ligninrepresents a major component of the terrestrial biomass and assumes agreat economic and ecological significance (Brown, 1985, J. Appl.Biochem. 7, 371-387; Whetten and Sederoff, 1991, Forest Ecology andManagement, 43, 301-316).

With regard to the exploitation of the biomass, first and foremost itshould be noted that lignin is a factor limiting the digestibility andnutritional yield of fodder crops. In fact, it is clearly demonstratedthat the digestibility of fodder crops by ruminants, is inverselyproportional to the lignin level of these plants, the nature of thelignins also being a determining factor in this phenomenon (Buxton andRoussel, 1988, Crop. Sci., 28, 553-558; Jung and Vogel, 1986, J. Anin.Sci., 62, 1703-1712).

Among the principle fodder crops in which it would be useful to reducethe level of lignins, there can be mentioned: alfalfa, fescue, maize,fodder used for silage . . . .

It should also be noted that high lignin levels are in part responsiblefor the limited quality of sunflower cakes intended for cattle food andthe reduction in the viability of certain seeds in the horticulturaldomain.

It can also be emphasized that the intense lignification which occursduring the storage of plant organs after harvest, rapidly renders cropssuch as asparagus, yams, carrots, etc. unfit for consumption.

Moreover, it should also be noted that more than 50 million tons oflignins are extracted from ligneous material each year in the productionof paper pulp in the paper industry. This extraction operation which isnecessary to obtain cellulose is costly in terms of energy andsecondarily polluting due to the chemical compounds employed in theextraction and which find their way into the environment (Dean &Eriksson, 1992, Holzforschung, 46, 135-147; Whetten and Sederoff, 1991,mentioned above).

To reduce the proportions of lignins (which depending on the speciesrepresent 20 to 30% of the dry material) to a few percent (2 to 5%)would represent an increase in yield, a substantial saving (chemicalproducts) and would contribute to the improvement of the environment(reduction in pollution). Given the scale on which ligneous material isused, these effects would have extremely significant repercussions. Inthis case, the species concerned could be the poplar, the eucalyptus,Acacia mangium, the Casuarina genus and all the angiosperms andgymnosperms used in the production of paper pulp.

In the two fields considered, it is clear that the reduction of ligninlevels must be moderate in order for the plant (or tree) to retain itscharacteristics of rigidity and its normal architecture, as the ligninswhich reinforce the cell walls play an important role in maintaining theupright habit of plants.

Natural variations in the levels of lignins observed in nature for thesame species (difference which can range up to 6-8% of the dry massbetween individuals) permit the reductions mentioned above.

The resistance to degradation of lignin, as well as the difficultiesencountered in its extraction, are probably due to the complex structureof this polymer constituted by ether and carbon--carbon bonds betweenthe monomers, as well as to the numerous chemical bonds existing betweenlignin and other components of the cell wall (Sarkanen and Ludwig, 1971,in Lignins: Occurrence, Formation, Structure and Reactions (K. V.Sarkanen and C. H. Ludwig eds) New York: Wiley--Interscience, pp. 1-18).

Starting from cinnamoyls-CoA, the biosynthesis of lignins in plants, iscarried out in the following manner: ##STR1##

One approach, using the genetic engineering route, to try to reduce thelevel of lignins in plants, would consist of inhibiting the synthesis ofone of the enzymes of the biosynthesis chain of these lignins indicatedabove.

A technique which is particularly appropriate within the scope of suchan approach, is that of using anti sense mRNA capable of hybridizingwith the mRNA coding for these enzymes, and as a consequence, ofinhibiting, at least partially, the production of these enzymes fromtheir corresponding mRNA.

Such an anti sense strategy, carried out using the gene coding for CADin tobacco, was the subject of the European Patent Application No. 584117, describing the use of anti sense mRNA capable of inhibiting theproduction of lignins in the plants by hybridizing with the mRNA codingfor CAD in these plants.

The results at the level of the plants transformed in this waydemonstrate a reduction in the activity of CAD, but paradoxically, thelignin levels show no change. Additional studies indicate that thelignins of the transformed plants are different from the controllignins, because the cinnamylic aldehydes are directly incorporated inthe lignin polymer.

DESCRIPTION OF THE INVENTION

One of the aims of the present invention is precisely that of providinga process allowing the lignin levels in plants to be effectivelyregulated, either in the sense of an appreciable reduction in theselevels relative to the normal levels in plants, or in the sense of anincrease in these levels.

Another aim of the present invention is to provide the tools for theimplementation of such a process, and more particularly usableconstructions for the transformation of plants.

Another aim of the present invention is to provide geneticallytransformed plants, in particular fodder crops capable of being moredigestible than untransformed plants, or also transformed plants ortrees for the production of paper pulp, and from which the extraction oflignin would be easier and less polluting than in the case ofuntransformed trees.

Another aim of the present invention is that of providing transformedplants which are more resistant to attack from the environment, inparticular parasitic attack, than untransformed plants are, or alsotransformed plants of a larger size, or of a smaller size (than that ofuntransformed plants).

A subject of the present invention is the use of recombinant nucleotidesequences containing one (or more) coding region(s) this (these)region(s) being constituted:

by a nucleotide sequence coding for a messenger RNA (mRNA), this mRNAitself coding for a cinnamoyl CoA reductase (CCR) in plants, or afragment of the nucleotide sequence mentioned above, this fragmentcoding for an mRNA, this mRNA itself coding for a fragment of a CCR inplants, this fragment of CCR having an enzymatic activity equivalent tothat of the CCR mentioned above, or a nucleotide sequence derived fromthe nucleotide sequence mentioned above, or derived from the fragmentmentioned above, in particular by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an mRNA, this mRNA itself coding for aderived protein having an enzymatic activity equivalent to that of theCCR mentioned above, or

by a nucleotide sequence complementary to all or part of the nucleotidesequence coding for an mRNA, or of a fragment of this sequence, or ofthe sequence derived from these last-named, as defined above, thiscomplementary sequence coding for an anti sense nucleotide sequence(also called anti sense mRNA) capable of hybridizing with one of themRNAs mentioned above,

for the transformation of plant cells with a view to obtainingtransgenic plants within which the biosynthesis of lignins is regulatedeither in the sense of an increase, or in the sense of a reduction inthe lignin levels produced, relative to the normal lignin levelsproduced in the plants.

By the expression "derived nucleotide sequence", in what has gone beforeand in what follows, is meant any sequence having at least about 50% ofnucleotides homologous with those of the sequence from which it isderived.

By "derived protein", in what has gone before and in what follows, ismeant any protein having at least about 50% of amino acids homologouswith those of the protein from which it is derived.

Among the nucleotide sequences capable of being used as coding regionsin the recombinant nucleotide sequences, there can principally bementioned:

the nucleotide sequence represented by SEQ ID NO 1, coding for an mRNA,this mRNA itself coding for the CCR of eucalyptus represented by SEQ IDNO 2,

the nucleotide sequence represented by SEQ ID NO 3, coding for an mRNA,this mRNA itself coding for the CCR of poplar represented by SEQ ID NO4,

the nucleotide sequence represented by SEQ ID NO 5, coding for an mRNA,this mRNA itself coding for the CCR of fescue represented by SEQ ID NO6,

the nucleotide sequence represented by SEQ ID NO 7, coding for an mRNA,this mRNA itself coding for the CCR of tobacco represented by SEQ ID NO8,

the nucleotide sequence complementary to that represented by SEQ ID NO1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7, this complementary sequencecoding for an anti sense mRNA capable of hybridizing with the mRNA codedby sequences SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 and SEQ ID NO 7respectively,

the nucleotide sequence derived from the sequence represented by SEQ IDNO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7, in particular by mutationand/or addition, and/or suppression, and/or substitution of one or morenucleotides, this derived sequence coding either for an mRNA itselfcoding for the CCR represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6or SEQ ID NO 8 respectively, or for a protein derived from theselast-named and having an enzymatic activity equivalent to that of saidCCRs in plants, in particular the nucleotide sequence derived fromsequence SEQ ID NO 1, and represented by SEQ ID NO 9, this latter codingfor an mRNA, this mRNA itself coding for a protein represented by SEQ IDNO 10 derived from the eucalyptus CCR mentioned above,

the nucleotide sequence derived from the complementary nucleotidesequence mentioned above, by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an anti sense mRNA capable of hybridizingwith one of the mRNAs mentioned above.

A more particular subject of the present invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence represented by SEQ ID NO 1, coding for an mRNA,this mRNA itself coding for the CCR represented by SEQ ID NO 2, or

a fragment of the nucleotide sequence mentioned above, this fragmentcoding for a CCR fragment represented by SEQ ID NO 2, this CCR fragmenthaving an enzymatic activity equivalent to that of the CCR mentionedabove, or

any nucleotide sequence derived from the sequence represented by SEQ IDNO 1 mentioned above, or from a fragment as described above of thissequence, in particular by mutation and/or addition, and/or suppression,and/or substitution of one or more nucleotides, this derived sequencecoding for an mRNA itself coding for the CCR represented by SEQ ID NO 2,or for a protein derived from the latter and having an enzymaticactivity equivalent to that of said CCR in plants, in particular thenucleotide sequence derived from the sequence SEQ ID NO 1, andrepresented by SEQ ID NO 9, this latter coding for an mRNA, this mRNAitself coding for a protein represented by SEQ ID NO 10 derived from theeucalyptus CCR mentioned above.

A more particular subject of the present invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence represented by SEQ ID NO 3, coding for an mRNA,this mRNA itself coding for the CCR represented by SEQ ID NO 4, or

a fragment of the nucleotide sequence mentioned above, this fragmentcoding for a CCR fragment represented by SEQ ID NO 4, this CCR fragmenthaving an enzymatic activity equivalent to that of the CCR mentionedabove, or

any nucleotide sequence derived from the sequence represented by SEQ IDNO 3 mentioned above, or from a fragment as described above of thissequence, in particular by mutation and/or addition, and/or suppression,and/or substitution of one or more nucleotides, this derived sequencecoding for an mRNA itself coding for the CCR represented by SEQ ID NO 4,or for a protein derived from the latter and having an enzymaticactivity equivalent to that of the said CCR in plants.

A more particular subject of the present invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence represented by SEQ ID NO 5, coding for an mRNA,this mRNA itself coding for the CCR represented by SEQ ID NO 6, or

a fragment of the nucleotide sequence mentioned above, this fragmentcoding for a CCR fragment represented by SEQ ID NO 6, this CCR fragmenthaving an enzymatic activity equivalent to that of the CCR mentionedabove, or

any nucleotide sequence derived from the sequence represented by SEQ IDNO 5 mentioned above, or from a fragment as described above of thissequence, in particular by mutation and/or addition, and/or suppression,and/or substitution of one or more nucleotides, this derived sequencecoding for an mRNA itself coding for the CCR represented by SEQ ID NO 6,or for a protein derived from the latter and having an enzymaticactivity equivalent to that of said CCR in plants.

A more particular subject of the present invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence represented by SEQ ID NO 7, coding for an mRNA,this mRNA itself coding for the CCR represented by SEQ ID NO 8, or

a fragment of the nucleotide sequence mentioned above, this fragmentcoding for a CCR fragment represented by SEQ ID NO 8, this CCR fragmenthaving an enzymatic activity equivalent to that of the CCR mentionedabove, or

any nucleotide sequence derived from the sequence represented by SEQ IDNO 7 mentioned above, or from a fragment as described above of thissequence, in particular by mutation and/or addition, and/or suppression,and/or substitution of one or more nucleotides, this derived sequencecoding for an mRNA itself coding for the CCR represented by SEQ ID NO 8,or for a protein derived from the latter and having an enzymaticactivity equivalent to that of said CCR in plants.

By protein having an enzymatic activity equivalent to that of CCRpresent in plants, and more particularly CCRs represented by SEQ ID NO2, SEQ ID NO 4, SEQ ID NO 6 and SEQ ID NO 8, is meant any protein havinga CCR activity as measured according to the method of Luderitz andGrisebach in Eur. J. Biochem. (1981), 119: 115-127.

By way of illustration, this method is carried out by spectrophotometricmeasurement of the reducing activity of the protein (CCR or derivative),by monitoring the disappearance of cinnamoyl CoA at 366 nm. The reactiontakes place at 30° C., for 2 to 10 minutes. The composition of thereaction medium is as follows: 100 mM phosphate buffer, pH 6.25, 0.1 mMNADPH, 70 μM Feruloyl CoA, 5 to 100 μl of enzymatic extract in a totalvolume of 500 μl.

Also a subject of the invention is any DNA sequence, characterized inthat it contains as coding region:

the nucleotide sequence complementary to that represented by SEQ ID NO1, this complementary sequence coding for an anti sense mRNA capable ofhybridizing with the mRNA itself coding for the CCR represented by SEQID NO 2, namely the mRNA coded by the sequence represented by SEQ ID NO1, or coded by a sequence derived from the latter, as defined above, or

a fragment of the complementary sequence mentioned above, this sequencefragment coding for an anti sense mRNA capable of hybridizing with themRNA itself coding for the CCR represented by SEQ ID NO 2, as definedabove, or

any nucleotide sequence derived from the complementary sequencementioned above, or from a fragment of this complementary sequence asdescribed above, in particular by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an anti sense mRNA capable of hybridizingwith the mRNA mentioned above, in particular the sequence complementaryto that represented by SEQ ID NO 9.

A more particular subject of the invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence complementary to that represented by SEQ ID NO3, this complementary sequence coding for an anti sense mRNA capable ofhybridizing with the mRNA itself coding for the CCR represented by SEQID NO 4, namely the mRNA coded by the sequence represented by SEQ ID NO3, or coded by a sequence derived from the latter, as defined above, or

a fragment of the complementary sequence mentioned above, this sequencefragment coding for an anti sense mRNA capable of hybridizing with themRNA itself coding for the CCR represented by SEQ ID NO 4, as definedabove, or

any nucleotide sequence derived from the complementary sequencementioned above, or from a fragment of this complementary sequence asdescribed above, in particular by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an anti sense mRNA capable of hybridizingwith the mRNA mentioned above.

A more particular subject of the invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence complementary to that represented by SEQ ID NO5, this complementary sequence coding for an anti sense mRNA capable ofhybridizing with the mRNA itself coding for the CCR represented by SEQID NO 6, namely the mRNA coded by the sequence represented by SEQ ID NO5, or coded by a sequence derived from the latter, as defined above, or

a fragment of the complementary sequence mentioned above, this sequencefragment coding for an anti sense mRNA capable of hybridizing with themRNA itself coding for the CCR represented by SEQ ID NO 5, as definedabove, or

any nucleotide sequence derived from the complementary sequencementioned above, or from a fragment of this complementary sequence asdescribed above, in particular by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an anti sense mRNA capable of hybridizingwith the mRNA mentioned above.

A more particular subject of the invention is any DNA sequence,characterized in that it contains as coding region:

the nucleotide sequence complementary to that represented by SEQ ID NO7, this complementary sequence coding for an anti sense mRNA capable ofhybridizing with the mRNA itself coding for the CCR represented by SEQID NO 8, namely the mRNA coded by the sequence represented by SEQ ID NO7, or coded by a sequence derived from the latter, as defined above, or

a fragment of the complementary sequence mentioned above, this sequencefragment coding for an anti sense mRNA capable of hybridizing with themRNA itself coding for the CCR represented by SEQ ID NO 7, as definedabove, or

any nucleotide sequence derived from the complementary sequencementioned above, or from a fragment of this complementary sequence asdescribed above, in particular by mutation and/or addition, and/orsuppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an anti sense mRNA capable of hybridizingwith the mRNA mentioned above.

It will be understood that the sequences represented by SEQ ID NO 1, SEQID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9, the complementarysequences, the derived sequences and the fragments of sequences of theinvention mentioned above, must be taken into consideration as beingrepresented in the 5'→3' direction.

Thus, the first nucleotide of a complementary sequence in the 5'→3'direction as described above, is the complement of the last nucleotideof the sequence in the 5'→3' direction coding for a CCR (or fragment ofCCR or derived protein), the second nucleotide of this complementarysequence is the complement of the next-to-last nucleotide of thesequence coding for a CCR, and so on, up to the last nucleotide of saidcomplementary sequence which is the complement of the first nucleotideof the sequence coding for a CCR.

The mRNA coded by the complementary sequence mentioned above is suchthat, when this mRNA is represented in the 5'→3' direction, its firstnucleotide corresponds to the last nucleotide of the sequence coding fora CCR, and therefore hybridizes with the last nucleotide of the mRNAcoded by the latter, while its last nucleotide corresponds to the firstnucleotide of the sequence coding for a CCR, and therefore hybridizeswith the first nucleotide of the mRNA coded by the latter.

In this way, by anti sense mRNA is meant in what has gone before and inwhat follows, any mRNA coded by foresaid complementary sequence andrepresented in the reverse direction (3'→5') to the direction in whichthe mRNA coded by the sequence coding for a CCR (or fragment of CCR orderived protein) is represented, this last-named mRNA still being called(5'→3') direction mRNA.

The term anti sense RNA therefore refers to an RNA sequencecomplementary to the sequence of bases of the messenger RNA, the termcomplementary having to be understood in this way that each base (or amajority of bases) of the anti sense sequence (read in the 3'→5'direction) is capable of pairing with the corresponding bases (G with C,A with U) of the messenger RNA (sequence read in the 5'→3' direction).

The anti sense RNA strategy, within the scope of the present invention,is a molecular approach particularly suitable to the objective ofmodifying the lignin levels of plants. The anti sense RNA is an RNAproduced by the transcription of a non-coding DNA strand (non sensestrand).

This anti sense strategy is more particularly described in the EuropeanPatent No. 240 208.

It is thought that the inhibition of the synthesis of a proteinaccording to the anti sense strategy, of CCR in the present case, is theconsequence of the formation of a duplex between the two complementaryRNAs (sense and anti sense) thus inhibiting the production of theprotein. However the mechanism remains unclear. The RNA--RNA complex mayinterfere either with a subsequent transcription, or with processing,transport or translation or even lead to degradation of the mRNA.

A combination of these effects is also possible.

The invention also relates to any mRNA coded by a DNA sequence accordingto the invention, and more particularly:

the mRNA coded by the DNA sequence represented by SEQ ID NO 1, or codedby a fragment or a derived sequence as defined above, said mRNA being inturn capable of coding for the CCR present in eucalyptus, as representedby SEQ ID NO 2, or for a fragment of this CCR or a derived protein asdefined above, in particular for the protein represented by SEQ ID NO10,

the mRNA coded by the DNA sequence represented by SEQ ID NO 3, or codedby a fragment or a derived sequence as defined above, said mRNA being inturn capable of coding for the CCR present in poplar, as represented bySEQ ID NO 4, or for a fragment of this CCR or a derived protein asdefined above,

the mRNA coded by the DNA sequence represented by SEQ ID NO 5, or codedby a fragment or a derived sequence as defined above, said mRNA being inturn capable of coding for the CCR present in fescue, as represented bySEQ ID NO 6, or for a fragment of this CCR or a derived protein asdefined above,

the mRNA coded by the DNA sequence represented by SEQ ID NO 7, or codedby a fragment or a derived sequence as defined above, said mRNA being inturn capable of coding for the CCR present in tobacco, as represented bySEQ ID NO 8, or for a fragment of this CCR or a derived protein asdefined above.

Also a subject of the invention is any anti sense mRNA, as definedabove, characterized in that it contains complementary nucleotides ofall or only part of the nucleotides constituting an mRNA as describedabove according to the invention, said anti sense mRNA being capable ofhybridizing (or pairing) with the latter.

As such, a more particular subject of the invention is the anti sensemRNAs coded by DNA sequences according to the invention, containing atleast one region of 50 bases homologous with those of a region ofcomplementary sequences of the above-mentioned DNA sequences of theinvention.

There is no upper size limit for the DNA sequences coding for an antisense RNA according to the invention; they can be as long as those ofthe messenger normally produced in these cells, even as long as thegenomic DNA sequence coding for the mRNA of the CCR.

Advantageously, such DNA sequences coding for an anti sense RNAaccording to the invention, contain between about 100 and about 1000base pairs.

A more particular subject of the invention is any anti sense sequencecontaining one (or more) anti sense mRNAs as described above, orfragment(s) of this (these) anti sense mRNA(s), and one (or more)sequence(s) corresponding to one (or more) catalytic domain(s) for aribozyme.

As such, a more particular subject of the invention is any anti sensesequence as described above, containing the catalytic domain for aribozyme flanked on either side by arms of about 8 complementary basesof sequences bordering a GUX unit (X representing C, U or A) containedin one of the mRNAs of the invention described above (also called targetRNAs) (Haseloff J., and Gerlach W. L., 1988, Nature, 334: 585-591).

The invention also relates to any DNA sequence capable of coding for ananti sense sequence as described above containing at least one catalyticdomain for a ribozyme related to one or more anti sense mRNAs of theinvention, or anti sense mRNA fragments (advantageously fragments ofabout 8 bases as described above).

A more particular subject of the invention is also any anti sensesequence containing:

any anti sense mRNA, as described above, characterized in that it iscoded by the nucleotide sequence complementary to that represented bySEQ ID NO 1, said anti sense mRNA being capable of hybridizing with themRNA coded by the DNA sequence represented by SEQ ID NO 1,

any anti sense mRNA, as described above, characterized in that it iscoded by the nucleotide sequence complementary to that represented bySEQ ID NO 3, said anti sense mRNA being capable of hybridizing with themRNA coded by the DNA sequence represented by SEQ ID NO 3,

any anti sense mRNA, as described above, characterized in that it iscoded by the nucleotide sequence complementary to that represented bySEQ ID NO 5, said anti sense mRNA being capable of hybridizing with themRNA coded by the DNA sequence represented by SEQ ID NO 5,

any anti sense mRNA, as described above, characterized in that it iscoded by the nucleotide sequence complementary to that represented bySEQ ID NO 7, said anti sense mRNA being capable of hybridizing with themRNA coded by the DNA sequence represented by SEQ ID NO 7,

any anti sense mRNA, as described above, characterized in that it iscoded by the nucleotide sequence complementary to that represented bySEQ ID NO 9, said anti sense mRNA being capable of hybridizing with themRNA coded by the DNA sequence represented by SEQ ID NO 9.

The invention also relates to the recombinant polypeptides coded the DNAsequences of the invention, said recombinant polypeptides having anenzymatic activity equivalent to that of the CCRs in plants, and moreparticularly the recombinant CCRs coded by the sequences represented bySEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 and SEQ ID NO 7, or by sequencesderived from these last-named according to the invention, in particularby SEQ ID NO 9.

A more particular subject of the invention is the recombinantpolypeptides, and in particular the recombinant CCRs, as obtained bytransformation of plant cells by integrating in a stable fashion arecombinant nucleotide sequence as defined above, containing a DNAsequence according to the invention, in their genome, in particularusing a vector as described hereafter.

By the expression "recombinant polypeptides", one must understand anymolecule having a polypeptide chain capable of being produced by geneticengineering, by means of a transcription phase of the DNA of thecorresponding gene, which leads to the obtaining of RNA which is thentransformed into mRNA (by suppression of the introns), the latter thenbeing translated by the ribosomes, in the form of proteins, all of thisbeing carried out under the control of appropriate regulation elementsinside a host cell. Consequently, the expression "recombinantpolypeptides" used does not exclude the possibility that saidpolypeptides contain other groups, such as glycosylated groups.

Of course, the term "recombinant" indicates that the polypeptide wasproduced by genetic engineering, as it results from the expression, inan appropriate host cell, of the corresponding nucleotide sequence whichhas been introduced beforehand into an expression vector used totransform said host cell. However, this term "recombinant" does notexclude the possibility that the polypeptide was produced by a differentprocess, for example by standard chemical synthesis according to knownmethods used for the synthesis of proteins, or by proteolytic cleavageof much larger molecules.

More particularly the invention relates to CCR such as is present ineucalyptus cells and represented by SEQ ID NO 2, the CCR such as ispresent in poplar cells and represented by SEQ ID NO 4, CCR such as ispresent in tall fescue cells and represented by SEQ ID NO 6, or CCR suchas is present in tobacco cells and represented by SEQ ID NO 8, or anyprotein derived from these last-named, in particular by addition, and/orsuppression, and/or substitution of one or more amino acids, inparticular the protein derived from eucalyptus CCR and represented bySEQ ID NO 10, or any fragment originating from said CCRs or from theirderived sequences, said fragments and derived sequences being capable ofhaving an enzymatic activity equivalent to that of the CCRs mentionedabove.

Also a subject of the invention is the nucleotide sequences coding forthe CCR represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO8 or SEQ ID NO 10, or any derived sequence or fragment of theselast-named, as defined above, said nucleotide sequences beingcharacterized in that they correspond to all or part of the sequencesrepresented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 or SEQID NO 9 respectively, or to any sequence derived from these last-namedby degeneration of the genetic code, and nevertheless being capable ofcoding for the CCRs or derived sequence or fragment of these last-named,as defined above.

A subject of the invention is also the complexes formed between the antisense mRNAs, as described above, and the mRNAs according to theinvention, capable of coding for all or part of a CCR in plants.

A more particular subject of the invention is the complex formed betweenthe mRNA coded by the sequence SEQ ID NO 1 and the anti sense mRNA codedby the sequence complementary to the sequence SEQ ID NO 1, the mRNAcoded by the sequence SEQ ID NO 3 and the anti sense mRNA coded by thesequence complementary to the sequence SEQ ID NO 3, the mRNA coded bythe sequence SEQ ID NO 5 and the anti sense mRNA coded by the sequencecomplementary to the sequence SEQ ID NO 5, the mRNA coded by thesequence SEQ ID NO 7 and the anti sense mRNA coded by the sequencecomplementary to the sequence SEQ ID NO 7, the mRNA coded by thesequence SEQ ID NO 9 and the anti sense mRNA coded by the sequencecomplementary to SEQ ID NO 9.

Also a subject of the invention is any recombinant nucleotide sequencecontaining one (or more) coding region(s), this (these) coding region(s)being constituted:

by a nucleotide sequence coding for an mRNA, this mRNA itself coding fora CCR in plants, or a fragment of the nucleotide sequence mentionedabove, this fragment coding for an mRNA, this mRNA itself coding for afragment of a CCR in plants, this fragment of CCR having an enzymaticactivity equivalent to that of the CCR mentioned above, or a nucleotidesequence derived from the nucleotide sequence mentioned above, orderived from the fragment mentioned above, in particular by mutationand/or addition, and/or suppression, and/or substitution of one or morenucleotides, this derived sequence coding for an mRNA, this mRNA itselfcoding for a derived protein having an enzymatic activity equivalent tothat of the CCR mentioned above, or

by a nucleotide sequence complementary to all or part of the nucleotidesequence coding for an mRNA, or of the fragment of this sequence, or ofthe sequence derived from these last-named, as defined above, thiscomplementary sequence coding for an anti sense mRNA capable ofhybridizing with one of the mRNAs mentioned above.

A more particular subject of the invention is any recombinant nucleotidesequence (or recombinant DNA), characterized in that it contains atleast one DNA sequence according to the invention, chosen from thosedescribed above, said DNA sequence being inserted in a heterologoussequence.

More particularly the invention relates to any recombinant nucleotidesequence as described above, containing, as coding region, thenucleotide sequence represented by SEQ ID NO 1, or by SEQ ID NO 3, or bySEQ ID NO 5, or by SEQ ID NO 7, or any fragment or a nucleotide sequencederived from the last-named, as defined above, said nucleotide sequencesor said fragment being inserted in a heterologous sequence, and beingcapable of coding for the CCR represented by SEQ ID NO 2, or by SEQ IDNO 4, or by SEQ ID NO 6 or by SEQ ID NO 8 respectively, or for afragment of these CCRs, or for a protein derived from these last-named,as defined above.

More particularly still the invention relates to any recombinantnucleotide sequence containing, as coding region, a nucleotide sequencecomplementary to that represented by SEQ ID NO 1, or by SEQ ID NO 3, orby SEQ ID NO 5, or by SEQ ID NO 7, or any fragment or any nucleotidesequence derived from this complementary sequence, as defined above,said complementary sequences or said fragment being inserted in aheterologous sequence, and being capable of coding for an anti sensemRNA capable of hybridizing with all or part of the mRNA coding for aCCR in plants, and more particularly with all or part of the mRNA codingfor the CCR represented by SEQ ID NO 2, or by SEQ ID NO 4, or by SEQ IDNO 6, or by SEQ ID NO 8 respectively.

The recombinant DNAs according to the invention are furthercharacterized in that they contain the elements necessary to regulatethe expression of the nucleotide sequence coding for a CCR, or of itscomplementary sequence coding for an anti sense mRNA according to theinvention, in particular a promoter and a terminator of thetranscription of these sequences.

Among the different promoters which are capable of being used in theconstruction of recombinant DNAs according to the invention, there canbe mentioned:

the endogenous promoter controlling the expression of CCR in a plant, inparticular the promoter situated upstream from the sequence representedby SEQ ID NO 1 in eucalyptus, or

constitutive-type strong-expression promoters, examples: ³⁵ S CAMV(described in Benfey et al. (1990), EMBO J., 9 (6), 1677-1684), EF1alpha(promoter of a gene with an elongation factor in the protein synthesisdescribed by Curie et al. (1991), Nucl. Acids Res., 19, 1305-1310),

promoters of specific type for particular expression in individualtissues, examples: CAD promoter (described by Feuillet C. (1993),University of Toulouse III Thesis), promoter GRP 1-8 (described byKeller and Baumgartner, (1991), Plant Cell., 3, 1051-1061) forexpression in specific vascular tissues.

The invention also relates to any recombinant nucleotide sequence asdescribed above, and also containing as coding region at least onenucleotide sequence coding for all or part of an mRNA itself coding foran enzyme other than CCR, which is implicated in a stage of thebiosynthesis of lignins in plants, in particular the mRNA coding forcinnamyl alcohol dehydrogenase (CAD), or also containing as codingregion at least one nucleotide sequence coding for all or part of ananti sense mRNA capable of hybridizing with the mRNA mentioned above, inparticular with the mRNA coding for CAD.

The recombinant nucleotide sequences of the invention mentioned aboveare advantageously obtained from vectors in which the DNA sequencescoding for an enzyme necessary for the biosynthesis of lignins in plantsare inserted.

By way of illustration, a more particular subject of the invention isthe vector called pEUCCR, containing the sequence represented by SEQ IDNO 1 cloned in the Bluescript vector, and deposited in culture in cellsof E. Coli DH5alpha at the Collection Nationale de Culture deMicro-organisms (CNCM) at the Institut Pasteur in Paris (France), onMar. 17, 1994 under No. I-1405.

The vectors mentioned above are digested using appropriate restrictionenzymes in order to recover said DNA sequences which are inserted inthem.

The last-named are then inserted downstream from an appropriatepromoter, and upstream from an appropriate terminator for theexpression, in the recombinant DNAs according to the invention.

More particularly a subject of the invention is the recombinant DNAscontaining the sequence represented by SEQ ID NO 1, coding for the mRNAitself coding for the CCR represented by SEQ ID NO 2, as obtained bydigestion of the pEUCCR vector mentioned above, recovery of the DNAsequence of the invention and insertion of the latter in the 5'→3'direction, in a heterologous DNA sequence containing a promoter and aterminator of the expression of said sequence.

A more particular subject of the invention is also the recombinant DNAscontaining the sequence complementary to the sequence SEQ ID NO 1,coding for an anti sense mRNA capable of hybridizing with the mRNAcoding for the CCR represented by SEQ ID NO 2, as obtained by digestionof the pEUCCR vector mentioned above, recovery of the DNA sequence ofthe invention, and insertion of the latter in the reverse direction,that is to say in the 3'→5' direction, in a heterologous DNA sequencecontaining a promoter and a terminator of the expression of thecomplementary sequence.

As an example of a terminator which can be used in such constructions,there can be mentioned the 3' end of the gene of the nopaline synthaseof Agrobacterium tumefaciens.

Therefore, in a general manner, the recombinant nucleotide sequencesaccording to the invention, containing a DNA sequence coding for a CCR(or a CCR fragment or derived protein), and/or other enzymes necessaryfor the biosynthesis of lignins, are obtained by recovery of said DNAsequence from the vectors mentioned above, and insertion of thissequence in the heterologous sequence, while the recombinant nucleotidesequences containing a DNA sequence coding for an anti sense mRNAaccording to the invention, are obtained by recovery of the DNA sequencementioned above and insertion of the latter in the reverse direction insaid heterologous sequence.

By way of illustration, there can be used all or part of thecomplementary DNA (cDNA) represented by SEQ ID NO 1, SEQ ID NO 3, SEQ IDNO 5, SEQ ID NO 7 or SEQ ID NO 9, for the construction of therecombinant DNAs mentioned above, or all or part of the genomic clonecorresponding to a CCR (which corresponds to the cDNAs mentioned above +possible introns). This genomic clone can be obtained using the cDNAs asprobes to screen a gene library, the latter being itself obtainedaccording to the method described by Sambrook, Fritsch and Maniatis,Molecular Cloning Laboratory Manual, Cold Spring Harbour LaboratoryPress, 1989.

A subject of the invention is also any recombinant vector, which can beused for the transformation of plants, characterized in that it containsa recombinant nucleotide sequence chosen from those described above,according to the invention, integrated in one of the sites of its genomewhich is not essential for its replication.

Among the recombinant vectors mentioned above which can be used for thetransformation of plants, there can be mentioned: the binary vectorsderived from pBIN 19 (Bevan et al., (1984), Nucl. Acids Res., 12(22),8711-8721).

Examples of the construction of recombinant vectors according to theinvention are described in the detailed description of the inventionwhich follows.

Also a subject of the present invention is a process for the regulationof the biosynthesis of lignins in plants, either by reducing, or byincreasing the quantities of lignin produced, relative to the normalquantities of lignins produced in these plants, said process containinga transformation stage of the cells of these plants using a vectorcontaining:

a nucleotide sequence coding for an mRNA, this mRNA itself coding for aCCR in plants, or a fragment of the nucleotide sequence mentioned above,this fragment coding for an mRNA, this mRNA itself coding for a fragmentof a CCR in the plants, this CCR fragment having an enzymatic activityequivalent to that of the CCR mentioned above, or a nucleotide sequencederived from the nucleotide sequence mentioned above, or derived fromthe fragment mentioned above, in particular by mutation and/or addition,and/or suppression, and/or substitution of one or more nucleotides, thisderived sequence coding for an mRNA, this mRNA itself coding for aderived protein having an enzymatic activity equivalent to that of theCCR mentioned above, or

a nucleotide sequence complementary to all or part of the nucleotidesequence coding for an mRNA, or of a fragment of this sequence, or ofthe sequence derived from these last-named, as defined above, thiscomplementary sequence coding for an anti sense mRNA capable ofhybridizing with one of the mRNAs mentioned above,

said transformation being carried out in particular using a vector asdescribed above.

A more particular subject of the invention is a process for thereduction of the quantity of lignins produced by biosynthesis in plants,this process being carried out by the transformation of the genome ofthese plants, by incorporating in it:

at least one DNA sequence according to the invention as described above,coding for an anti sense mRNA capable of hybridizing with all or part ofthe mRNA coding for the CCR represented by SEQ ID NO 2, SEQ ID NO 4, SEQID NO 6 or SEQ ID NO 8, or for a protein derived from these last-namedas defined above, in particular for the protein represented by SEQ ID NO10,

and, if appropriate, at least one DNA sequence coding for an anti sensemRNA capable of hybridizing with an mRNA coding for an enzyme other thanCCR, which is implicated in a stage of the biosynthesis of lignins inplants, in particular the mRNA coding for CAD,

said transformation being carried out:

either using a recombinant vector as described above, containing a DNAsequence coding for an anti sense mRNA capable of hybridizing with themRNA coding for CCR or for a derived protein, as defined above, and ifappropriate, containing one or more DNA sequence(s) coding for an antisense mRNA capable of hybridizing with an mRNA coding for an enzymeother than CCR as defined above,

or using several recombinant vectors at least one of which contains aDNA sequence coding for an anti sense mRNA capable of hybridizing withthe mRNA coding for CCR or for a derived protein, as defined above,while the other recombinant vector(s) contain(s) a DNA sequence codingfor an anti sense mRNA capable of hybridizing with an mRNA coding for anenzyme other than CCR, as defined above.

Another process for reducing the quantity of lignins produced bybiosynthesis in plants, is that carried out by transformation of thegenome of these plants, by incorporating in it:

at least one DNA sequence according to the invention represented by SEQID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7, or a fragment or asequence derived from the latter, as defined above, in particular thesequence represented by SEQ ID NO 9,

and, if appropriate, at least one DNA sequence coding for all or part ofan enzyme other than CCR, which is implicated in a stage of thebiosynthesis of lignins in plants, in particular a DNA sequence codingfor all or part of CAD,

said transformation being carried out:

either using a recombinant vector as described above, containing the DNAsequence according to the invention mentioned above, or a fragment or asequence derived from the latter, as defined above, and, if appropriate,containing one or more DNA sequence(s) coding for all or part of anenzyme other than CCR, as defined above,

or using several recombinant vectors at least one of which contains aDNA sequence according to the invention mentioned above, or a fragmentor a sequence derived from the latter, as defined above, while the otherrecombinant vector(s) contain(s) a DNA sequence coding for all or partof an enzyme other than CCR as defined above.

This last method requires a co-suppression mechanism. Co-suppression wasobserved when copies of the endogenous gene were introduced into thegenome. Whilst the co-suppression mechanism is at present unknown, oneof the most frequently accepted hypotheses is that the negativeregulation of the expression of the gene may result from the productiona small quantity of anti sense RNA derived from a transgene through areading of the "wrong" strand of the transgene (Grierson et al., TrendsBiotech., 9: 122-123).

A subject of the invention is also a process for the reduction of thequantity of lignins produced by biosynthesis in plants, this processbeing carried out by transformation of the genome of these plants byincorporating in it a DNA sequence as described above according to theinvention, coding for an anti sense sequence containing one or (more)catalytic domain(s) for a ribozyme related to one (more) anti sensemRNAs, or anti sense mRNA fragments of the invention, saidtransformation being carried out using a recombinant vector containing arecombinant nucleotide sequence according to the invention itselfcontaining the DNA sequence mentioned above.

It is important to note that the methods mentioned above allowtransformed plants to be produced having different level of reduction inthe CCR activity (according to the level of insertion of the DNAsequence coding for the anti sense mRNA, the number of copies of thisDNA sequence integrated in the genome . . . ) and therefore the ligninlevels.

The choice of transformants will therefore allow a controlled modulationof the lignin levels compatible with a normal development of the plant.

In a general manner, if one considers that the normal average level oflignins of a plant varies between about 15% and about 35% by weight ofdry matter, the reduction in the level of lignins resulting from theimplementation of one of the processes mentioned above, isadvantageously such that the plants transformed in this way have anaverage level of lignins varying between about 10% and about 30%, oralso between about 12% and about 32%.

By way of illustration, the lignin level of a plant can be measuredaccording to a variant of the method of Johnson et al., (1961),T.A.P.P.I., 44, 793-798, which is described in detail in Alibert andBoudet (1979), Physiol., Veg., 17 (1), 67-74, and the principal stagesof which are the following: after obtaining a benzene alcohol powdercontaining lignins of plant material, the lignins are solubilized withacetyl bromide and analyzed as a function of their absorption in theultraviolet.

A more particular subject of the invention is the use of the processesmentioned above for the reduction of lignin levels in plants, forobtaining genetically-transformed fodder crops, having lignin levelswhich are reduced relative to the normal lignin levels in these plants,and in this way the digestibility of which is improved relative to thesame untransformed plants.

Among the main fodder crops capable of being transformed within thescope of the present invention, there can be mentioned: alfalfa, fescue,maize used for silage, etc . . . .

The invention also relates to the use of the processes mentioned abovefor the reduction of the lignin levels in plants, for obtaininggenetically-transformed plants, and more particularly trees, havinglignin levels which are reduced relative to the normal lignin levels inthese plants, these plants or trees being particularly advantageous foruse within the scope of the production of paper pulp.

A third potential field of use of the processes mentioned above for thenegative regulation of the expression of the CCR gene relates to thestimulation of the growth of transformed plants. Various argumentsemphasize (Sauter and Kende, 1992, Plant and Cell Physiology, 33(8):1089), that early and rapid lignification acts as a brake on cellenlargement and therefore on the growth of plants. Thus theimplementation of the processes mentioned above is capable of allowingplants transformed in this way with reduced lignification better growthand therefore better yields.

The invention also relates to a process for increasing the quantity oflignins produced by biosynthesis in plants, this process being carriedout by transformation of the genome of these plants, by incorporating init:

at least one DNA sequence according to the invention represented by SEQID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7, or a fragment or asequence derived from the latter, as defined above, in particular thesequence represented by SEQ ID NO 9,

and, if appropriate, at least one DNA sequence coding for all or part ofan enzyme other than CCR, which is implicated in a stage of thebiosynthesis of lignins in plants, in particular a DNA sequence codingfor all or part of CAD,

said transformation being carried out:

either using a recombinant vector as described above, containing the DNAsequence according to the invention mentioned above, or a fragment or asequence derived from the latter, as defined above, and, if appropriate,containing one or more DNA sequence(s) coding for all or part of anenzyme other than CCR, as defined above,

or using several recombinant vectors at least one of which contains aDNA sequence according to the invention mentioned above, or a fragmentor a sequence derived from the latter, as defined above, while the otherrecombinant vector(s) contain(s) a DNA sequence coding for all or partof an enzyme other than CCR as defined above.

In a general manner, still if one considers that the normal averagelignin level of a plant varies between about 15% and about 35% by weightof dry matter, the increase in the lignin level resulting from theimplementation of the process mentioned above, is advantageously suchthat the plants transformed in this way have an average lignin levelvarying between about 20% and about 40%, between about 18% and about38%.

A more particular subject of the invention is the use of the processmentioned above for the increase in lignin levels in plants (also calledprocess for the overexpression of the CCR gene), to obtain geneticallytransformed plants, having increased lignin levels relative to thenormal lignin levels in plants, and of which the resistance propertiesto environmental attacks, in particular to parasite attacks, are thusimproved relative to the same untransformed plants. It is particularlyadvantageous in this last case to use in combination with the CCR gene,or a derived sequence, in the vectors mentioned above, specificpromoters particularly expressed in the surface tissues and/or inresponse to injury.

Furthermore, the invention also relates to the use of the processmentioned above for the overexpression of the CCR gene, to improve thegrowth of plants genetically transformed in this way, in particular incertain fields such as horticulture or arboriculture, where theobtaining of small plants is desired.

Finally, the benzene rings in lignin have higher intrinsic energy thanthe aliphatic chains of the glucose residues of cellulose. Therefore,the increase in the proportion of lignins in plants used as fuels,according to the process of the invention mentioned above, allowsimprovement of the potential energy of these fuel plants transformed inthis way.

In the two cases of negative regulation or overexpression of CCR, it isquite conceivable that the modulation of this activity has repercussionson the lignin levels of the transformed plants. In fact, the CCR, theactivity level of which is very low in the plant, appears to constitutethe regulating enzyme for the synthesis of lignins.

Regarding the transformation techniques used for the implementation ofone of the process described above of the invention, the followingtechniques will be advantageously used:

A) The technique of transformation using plasmid Ti of Agrobacteriumtumefaciens described by Bevan (1984) Nucleic Acid Research, 12:8711-8721. It essentially uses the co-culture method, and brings intoplay a co-transformation with a selection gene in order to be able tolocate the transformants.

It is particularly applicable to dicotyledons, ex.: tobacco, alfalfa,oilseed rape.

B) The technique of direct transfer of genes by biological ballisticsdescribed in detail by (Zumbrum et al., 1989, Technique 1, 204-216;Sanford et al., 1991, Technique 3, 3-16).

This technique involves the combination of the recombinant DNA accordingto the invention with microparticles of gold or tungsten which arepropelled using a gene gun onto the tissue to be transformed. It will beparticularly applied to the transformation of refractory species toagrobacteria.

In the two cases mentioned above, verification of the presence of therecombinant DNA according to the invention will be carried out usingsouthern type hybridization and genetic amplification (polymerase chainreaction) tests, using oligonucleotide probes and primers originating inparticular from the sequence SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQID NO 7 or SEQ ID NO 9.

The invention also relates to the plant cells transformed by a vectoraccording to the invention, in particular using the techniques describedabove, and containing a DNA sequence according to the inventionintegrated in a stable fashion in their genome.

Also a subject of the invention is the transformed plants as obtained byculturing the transformed cells mentioned above.

The transformed plants can then be propagated by sexual route or byvegetative route in vitro or in natura.

A subject of the invention is also the fragments of plants, inparticular fruits, seeds, pollen, transformed by incorporation in theirgenome of a DNA sequence according to the invention, using therecombinant vectors mentioned above.

The invention also relates to the antibodies directed against therecombinant polypeptides of the invention, and more particularly thosedirected against the recombinant CCRs mentioned above.

Such antibodies can be obtained by the immunization of an animal withthese polypeptides followed by recovery of the antibodies formed.

It will be understood that this production is not limited to polyclonalantibodies.

It also applies to any monoclonal antibody produced by any hybridomacapable of being formed, by standard methods, from the splenic cells ofan animal, in particular mice or rats, immunized against one of thepurified polypeptides of the invention on the one hand, and cells of anappropriate myeloma on the other hand, and of being selected, by itscapacity to produce monoclonal antibodies recognising the polypeptidementioned above initially used for the immunization of the animals.

Also a subject of the invention is the use of antibodies mentioned abovedirected against the recombinant polypeptides of the invention, for theimplementation of a detection or analysis method of CCRs in plants,starting from samples taken from these last-named.

More details of the invention will be given in the description whichfollows for the obtaining of CCR in purified form from the eucalyptus,and of the cDNA coding for CCR of the eucalyptus, poplar, tall fescueand tobacco.

EXAMPLES

Although the invention has been described in conjunction with specificembodiments, it is evident that many alternatives and variations will beapparent to those skilled in the art in light of the foregoingdescription. Accordingly, the invention is intended to embrace all ofthe alternatives and variations that fall within the spirit and scope ofthe appended claims.

All references cited through the disclosure are hereby incorporated byreference.

A) Obtaining purified eucalyptus CCR and the cDNA coding for aeucalyptus CCR.

I Purification of eucalyptus CCR.

CCR has been the subject of a number of very restricted studies. Amongthe few publications relating to it, there can be mentioned:

Wengenmayer H., Ebel J., Grisebach H., 1976--Enzymatic synthesis oflignin precursors, purification and properties of a cinnamoyl CoA: NaDPHreductase from cell suspension cultures from soyabean (Glycine max),Eur. J. Biochem., 65: 529-536.

Luderitz T., Grisebach H., 1981--Enzymatic synthesis of ligninprecursors, comparison of cinnamoyl: CoA reductase and cinnamyl alcoholdehydrogenase: NADP dehydrogenase from spruce (Picea abies L.) andsoyabean (Glycine max L.), Eur. J. Biochem., 119: 115-127.

Sarni F., Grand C., Boudet A. M., 1984--Purification and properties ofcinnamoyl: CoA reductase and cinnamyl alcohol dehydrogenase from poplarstems (Populus x euramericana). Eur. J. Biochem., 139: 259-265.

The work described hereafter has contributed to the definition of aprotocol for the original, simple and rapid purification of eucalyptusCCR. This protocol is also more effective than those describedpreviously in the literature. In fact, it has allowed for the firsttime, quantities of homogeneous purified enzyme to be obtained which aresufficient to obtain internal peptide sequences and eventually lead tothe cloning of the corresponding cDNA.

All the purification stages of the CCR were carried out at 4° C.

1. Obtaining a crude extract of eucalyptus xylem.

The plant matter was obtained by "scraping" a xylem-enriched tissuefraction from branches of 5-year-old Eucalyptus gunnii.

300 g of xylem frozen beforehand in liquid nitrogen was reduced topowder using a coffee mill. The homogenate thus obtained was homogenizedin one liter of extraction buffer (100 mM Tris-HCl pH 7.6, 2% PEG 6000,5 mM DTT, 2% PVPP), filtered through two thicknesses of Miracloth, andbrought to 30% saturation with ammonium sulphate. After centrifuging for30 minutes at 15000× g, the pellet obtained is resuspended in 60 ml ofbuffer 1 [20 mM Tris-HCl pH 7.5, 5 mM DTT (dithiothreitol), 5% ethyleneglycol]. The extract thus obtained is "clarified" by a 15 minutecentrifugation at 10000× g, then desalted by passage through a SephadexG25 equilibrated with buffer 1.

2. Affinity chromatography on Red Sepharose.

The crude desalted extract is applied to a "Red Sepharose" affinitycolumn (1.5×19 cm, Pharmacia), equilibrated with buffer 1. After aninitial rinsing of the column with 50 ml of buffer 1, the proteins areeluted with a linear gradient of Tris from 20 mM to 1.5 M Tris-HCl pH7.5, containing 5 mM DTT, 5% ethylene glycol. The total volume of thegradient is 200 ml and the flow rate is 36 ml/hr. The fractions having aCCR activity are collected and desalted by passage through a SephadexG25 column, equilibrated with buffer 1.

3. Anion exchange chromatography on MonoQ.

The fractions collected and desalted in this way are chromatographed ona MonoQ anion exchange column (HR 5/5, Pharmacia). Elution of theproteins is carried out by the application of a linear gradient of 20 to300 mM Tris-HCl pH 7.5, containing 5% ethylene glycol and 5 mM DTT. Thetotal volume of the gradient is 50 ml and the flow rate is 1 ml/min. Aswith the previous stage, the fractions containing the active CCR enzymeare collected and desalted, but in this case the equilibration bufferfor the Sephadex G25 columns is a 20 mM phosphate buffer, pH 7.6,containing 5 mM DTT (buffer 2).

4. Affinity chromatography on "Mimetic Red".

The group of CCR fractions obtained in this way is applied to a MimeticRed 2 A6XL column (ACL, Cambridge). The column is washed beforehand with30 ml of buffer 2 containing 8 mM of NAD. The object of this washing isto eliminate enzymes functioning specifically with NAD as a co-factor,such as the malate dehydrogenase which copurifies with the CCR in theprevious stages. The specific elution of CCR is obtained by applicationof a gradient (15 ml) of NADP 0-8 mM in buffer 2. The fractionscontaining the pure and active CCR are stored at -80° C. after theaddition of a stabiliser (ethylene glycol at a final concentration of5%).

The purified enzyme thus obtained has a specific activity of 451 nKat/mgof protein, using feruloyl CoA as substrate. The yield obtained (36 μgof pure protein for 300 g of initial plant matter) does not reflect theproportion of CCR in planta, in fact due to a major concern to eliminatethe maximum amount of contaminants at each purification stage, only thefractions having a very strong CCR activity are treated in the followingstage. The purification factor obtained with this protocol is 282.

II Characterisation of the CCR.

The eucalyptus CCR is a monomer of 38 kD as witnessed by the convergentresults obtained for the size of the native enzyme by exclusionchromatography on Superose 6 (Pharmacia), and for the size of themonomer sub-unit on denaturing electrophoresis gel. The isoelectricpoint, estimated by chromatography on MonoP (Pharmacia) is close to 7.

Research into the pH and optimum buffer shows that the measurement ofCCR activity as it was initially described (Luderitz and Grisebach,1981), is perfectly adapted to the measurement of the eucalyptus CCRactivity (100 mM phosphate buffer, pH 6.25).

The purity of the CCR, present in the state of a single band onone-dimensional electrophoresis gel (SDS PAGE) was confirmed by theobtaining of a single spot after two-dimensional electrophoresis andstaining with silver salts.

III Obtaining the cDNA coding for eucalyptus CCR

In order to avoid a possible problem of non-detectable residualcontamination, the pure enzyme was subjected to a preparativeelectrophoresis under semi-denaturing conditions and digested in situ inthe gel. The digestion was carried out using endolysine C whichspecifically cuts the proteins after the lysine residues, allowingrelatively long peptides to be obtained. The peptides resulting from thedigestion were separated by reversed phase HPLC and sone of them weresequenced using a protein microsequencer (Applied Biosystems 470). Thesequences of these internal peptides are shown below:

    peptide 8                                                                                 (a)  Asn-Trp-Tyr-Cys-Tyr-Gly-Lys                                              (b)  His-Leu-Pro-Val-Pro-X-Pro-Pro-Glu-Asp                                         Ser-Val-Arg                                                         X representing any amino acid                                          peptide 10                                                                           Thr-Tyr-Ala-Asn-Ser-Val-Gln-Ala-Tyr-Val-His-                                  Val-Lys                                                                peptide 13                                                                           Gly-Cys-Asp-Gly-Val-Val-His-Thr-Ala-Ser-Pro-                                  Val-Thr-Asp-Asp                                                        #peptide 17                                                                          Leu-Arg-Asp-Leu-Gly-Leu-Glu-Phe-Thr-Pro-Val-                                  Lys                                                                    peptide 18                                                                           Gly-Asp-Leu-Met-Asp-Tyr-Gly-Ser-Leu-Glu-Glu-                                  Ala-Ile-Lys                                                        

The cDNA coding for the CCR was obtained by screening usingoligonucleotides from a cDNA library constructed in the λZAPII phage(commercially available vector, Stratagene) from messenger extracts ofxylem of Eucalyptus gunnii. 600,000 phages were screened using a groupof degenerate oligonucleotides labelled at the 3' end with phosphorus32, using a terminal transferase. The sequences of the oligonucleotidesused for the screening were determined from the internal peptidesequences mentioned above. These peptides having been generated bycutting with endolysine C, a lysine was again added in first position inorder to allow the production of oligonucleotides with the leastdegeneration. In fact, this amino acid which can only be coded by twocodons, is part of the amino acids whose code is least degenerate and asa consequence is quite suitable for the production of oligonucleotidesfrom peptide sequences.

The sequences of oligonucleotides used for the screening of theeucalyptus cDNA library derived from the underlined amino acids(I-inosine) are indicated hereafter:

    peptide 8 (a)                                                                             Lys-Asn-Trp-Cys-Tyr-Gly-Lys                                       oligonucleotide 8                                                                         AA(A/G)AA(C/T)TGGTA(C/T)TG(C/T)TA(T/C)GG                                      IAA                                                               peptide 13  Lys-Gly-Cys-Asp-Gly-Val-Val-His-Thr-Ala-                                      Ser-Pro-Val-Thr-Asp-Asp                                           oligonucleotide 13                                                                        AA(G/A)GGITG(C/T)GA(C/T)GGIGTIGTICA                               peptide 17  Lys-Leu-Arg-Asp-Leu-Gly-Leu-Glu-Phe-Thr                                       Pro-Val-Lys                                                       oligonucleotide 17                                                                        GA(G/A)TT(C/T)ACICCIGTIAA                                         peptide 18  Lys-Gly-Asp-Leu-Met-Asp-Tyr-Gly-Ser-Leu-                                      Glu-Glu-Ala-Ile-Lys                                               oligonucleotide 18                                                                        AA(G/A)GGIGA(C/T)(C/T)TIATGGA(C/T)TA                                          (C/T)GG                                                       

The hybridization conditions used for the screening are as follows: thepre-hybridization is carried out for 6 to 7 hours in 5× SSPE, 0.25%skimmed milk powder, 0.05% SDS (sodium dodecyl sulphate) at 42° C. Thehybridization is carried out in the same solution, in the presence of 4oligonucleotides labelled at the 3' end with ddATPα³² P, for 24 hours at42° C. At the end of these 24 hours of hybridization, the filters arewashed three times for 15 minutes in 2× SSC, 0.1% SDS then put incontact with an autoradiographic film for 24 hours at -80° C. The phageshybridizing with the group of oligonucleotides were purified by twoadditional screening rounds (plaque purification). Once purified the sixpositive clones were tested with each of the oligonucleotides takenindependently. One phage reacted positively with the 4 oligonucleotides,it was treated in such a manner as to "excise" the recombinantBluescript plasmid by following the manufacturer's instructions(Stratagene). The restriction map of the insert (coding for CCR)contained in this plasmid is schematized in FIG. 1.

IV Characterisation and identification of the cDNA of CCR

The amino acid sequence (represented by SEQ ID NO 2) derived from thenucleotide sequence (represented by SEQ ID NO 1) codes for a protein of335 amino acids the molecular weight of which is 36.5 kD and theisoelectric point of which is about 5.8. It is important to emphasizethat all the peptide sequences obtained from the purified CCR are foundagain in the peptide sequence derived from the nucleotide sequence ofthe cDNA.

Searches for homologies with clones which already exist were carried outusing BLAST and FASTA programs in all available protein and nucleiclibraries. One significant homology was found with another reductase ofthe metabolism of phenol compounds, dihydroflavonol reductase (DFR). Theidentity is about 40% and the similarity close to 20% between thepeptide sequence derived from the cDNA of the CCR and the sequences ofthe various dihydroflavonol reductase itemized in the libraries, whichconfirms that the identified clone is different from a clone coding fora DFR.

V Production of active recombinant CCR in E. Coli.

In order to proceed further with the identification of the cDNA of CCR,the recombinant protein was produced in E. Coli and its enzymaticactivity was researched. The experimental details of this approach aredescribed hereafter.

1--Introduction of the cDNA in expression vector pT7-7.

In order to be able to clone the cDNA in the expression vector pT7-7(commercially available), under the control of a promoter of T7polymerase, we had to introduce a Ndel site in the ATG position of thecDNA. This was carried out using a Taq polymerase during a geneamplification reaction by PCR (Polymerase Chain Reaction) between amutated oligonucleotide and a commercial primer, T7, situated on Bluescript downstream from the 3' end of the cDNA. The amplification productobtained is digested by Kpnl, this site is then repaired using a klenowfragment of DNA polymerase I before subjecting the fragment to digestionby Ndel, then the fragment obtained containing an Ndel site in the 5'position and a blunt end in the 3' position is inserted using a DNA T4ligase in the vector pT7-7 which has been opened beforehand by Ndel andSmal.

The sequence of the mutated oligonucleotide mentioned above is indicatedhereafter.

The underlined and italicized bases were modified relative to theinitial sequence allowing the creation of a Ndel site (CATATG):

5'GGCAATCCCCATATGCCCGTCGACGC3'

2. Overexpression of CCR in E. Coli BL21

The construction thus obtained is introduced into the strain E. ColiBL21 (commercially available) which carries on its chromosome the geneof the T7 polymerase under the control of promoter lac UV5, a promoterinducible by IPTG. The recombinant culture is cultivated at 37° C. untilan OD measured at 1 to 600 nm is obtained, then the production of CCR isinduced by the addition of IPTG (0.25% final) into the culture medium.Samples are removed at different times after the induction and the cellsare lyzed according to the protocol described by Grima-Pettenati et al.(1993). After centrifuging, the supernatant containing the solubleproteins is used to measure the CCR activity and to show the productionof CCR after electrophoresis under denaturing conditions. In FIG. 2, theappearance of a polypeptide of about 38 kD should be noted, theintensity of which increases with post-induction time and which does notexist in the negative controls (strain BL21 containing only the vectorpT7-7 without insert). Moreover, the final proof of the identity of theCCR clone is provided by measurement of the CCR activity (about 7nKat/ml of culture after 3 hours of induction at 37° C.) in the proteinextracts originating from BL21 strains containing pT7-7+CCR cDNA only.

Annotations of figures:

FIG. 1: restriction map of cDNA coding for eucalyptus CCR.

FIG. 2: disclosure of an electrophoresis gel with Coomassie blue underdenaturing conditions (SDS-PAGE) of total protein extracts of E. ColiBL21 containing the vector pT7-7 without insert (track 6 at time 0 ofthe induction by IPTG; 7, 3 hours after induction) and the vector pT7-7containing the cDNA of the CCR (tracks 1, 2, 3, 4, 5, respectively, time0 of the induction, 30 min, 1, 2, 3 hours after induction), the arrowdesignates the CCR monomer.

FIG. 3: schematic representation of plasmid pEUCCR containing thesequence represented by SEQ ID NO 1 (and identified by CCR in theplasmid pEUCCR)

FIG. 4: schematic representation of the construction of a vectorcontaining a DNA sequence coding for eucalyptus CCR according to theinvention (or sense CCR vector).

FIG. 5: schematic representation of the construction of a vectorcontaining a DNA sequence coding for an anti sense RNA capable ofhybridizing with the mRNA coding for eucalyptus CCR according to theinvention (or anti sense CCR vector).

Regarding the DNA source used for the construction of an anti sense (orsense) vector

The anti sense RNA is preferably derived from the sequence contained inthe clone pEUCCR. This sequence can be obtained in different ways:

1) by cutting the DNA sequence (cDNA) of the CCR contained in pEUCCRwith appropriate restriction enzymes,

2) by carrying out a gene amplification (PCR) using oligonucleotidesdefined so as to synthesis the desired DNA fragment.

The DNA fragment thus obtained is cloned in a plant expression vectordownstream from a promoter and upstream from a terminator. The cloningis carried out in such a way that the DNA fragment is inserted in thereverse orientation to the promoter. In this new vector, the strandwhich was initially the template strand becomes the coding strand andvice versa.

The new vector codes for an RNA the sequence of which is complementaryto the sequence of the messenger RNA derived from the sequence containedin pEUCCR.

Therefore the 2 RNAs are complementary not only by their sequence butalso by their orientation (5'-3')

As a source of the DNA for the transcription of the anti sense RNA it ispractical to use a cDNA clone such as that contained in pEUCCR.

Example of cloning (cf. FIG. 5)

The cDNA of CCR is obtained by a double digestion (BamHI and KpnI) fromthe vector pEUCCR. The DNA fragment thus released is physicallyseparated from the cloning vector by agarose gel electrophoresis(Bluescript).

The part of the gel containing this DNA fragment is cut out and treatedin such a way as to obtain the purified DNA (several methods may be usedsuch as "Low melting Agarose", described in Sambrook et al. mentionedabove, the Gene Clean, the kit of which is commercially available).

The fragment carrying the BamHI and KpnI ends is "ligated" with anexpression vector of plants digested beforehand by these same enzymeschosen in such a way that the cDNA is inserted in the reverseorientation relative to the 35S promoter. The strand which will betranscribed in the plants will in this case be the non-coding strand.

Example of sense cloning (cf. FIG. 4)

In this case "practical" restriction sites do not exist to carry outtranslational fusion with the ³⁵ S promoter of the expression vector.New more convenient sites were inserted using the gene amplificationtechnique (PCR). Two oligonucleotides were defined in position 5' and 3'of the cDNAs to which the sequences of sites recognised by KpnI andBamHI were added (NB.: these are the same sites which were used for theanti sense cloning mentioned above, but positioned differently relativeto the 5'-3' orientation).

The gene amplification leads to a fragment being obtained containing allthe sequence coding for the cDNA flanked by 2 restriction sites. Therest of the procedure is identical to that described for the anti senseconstruction.

But, in this case, a fusion of the promoter in phase with the ATG of theCCR is carried out which must lead to an overexpression of messenger RNAand therefore of CCR protein.

B) Obtaining the cDNA coding for poplar CCR.

The cDNA was obtained by screening a cDNA library constructed in theλZAPII phage (Stratagene) from messenger extracts of xylem of 2-year-oldpoplars (Populus trichocarpa). The phages were screened using cDNAisolated from Eucalyptus gunnii (fragment Xhol of 804 bp of pEUCCR)labelled by random priming with dCTPα³² P.

The hybridization conditions used for the screening are as follows:

the pre-hybridization is carried out for one night at 68° C. in 6× SSC,5× Denhardt, 0.5% SDS, 100 μg of salmon sperm DNA. The hybridization iscarried out in the same buffer without the addition of the Denhardtreagent, for one night at 60° C. The membranes are then washed twice for30 minutes in 0.1× SSC, 0.5% SDS at 60° C.

One clone containing the complete cDNA was purified, sub-cloned andsequenced in both directions. The cDNA sequence is that represented bySEQ ID NO 3 and the derived amino acid sequence of the latter is thatrepresented by SEQ ID NO 4.

Comparison between the amino acid sequences of the eucalyptus CCR andthat of the poplar show an identity of 83% and a homology of 93% betweenthese two sequences.

C) Obtaining the cDNA coding for fescue CCR.

The cDNA was obtained by screening a cDNA library constructed in theλZAPII phage (Stratagene) from messenger extracts of the leaves ofFetusca arundinacea. 500,000 phages were screened using cDNA isolatedfrom Eucalyptus gunnii (fragment Xhol of 804 bp of pEUCCR) labelled byrandom priming with dCTPα³² P.

The hybridization conditions used for the screening are as follows:

the pre-hybridization is carried out for 6 hours in 5× SSPE, SDS 0.5%,salmon sperm DNA 0.1 mg/ml, Ficoll type 400 1 mg/ml,polyvinylpyrrolidone 1 mg/ml, BSA 1 mg/ml at 60° C. The hybridization iscarried out in the same solution in the presence of the probe labelledwith dCTPα³² P, for 16 hours at 60° C. The filters are then washed twicefor 15 minutes in 2× SPPE, 0.1% SDS at 60° C. then twice in 0.5× SSPE,0.1% SDS at 60° C. and finally put in contact with an autoradiographicfilm for 3 days at -80° C. The phages were purified by 2 or 3 additionalscreening rounds.

Once purified, 3 clones were analyzed after the plasmid pBluescript hadbeen excised according to the manufacturer's instructions (Stratagene).

The sequencing allowed one clone to be eliminated and the other 2 clonesto be shown to be identical and to correspond to the nucleotide sequencerepresented by SEQ ID NO 5 coding for the fescue CCR as represented bySEQ ID NO 6.

D) Obtaining the cDNA coding for tobacco CCR.

The cDNA was obtained by screening a cDNA library constructed in theλZAPII phage (Stratagene) from messenger extracts of tobacco stems. Thephages were screened using cDNA isolated from Eucalyptus gunnii(fragment Xhol of 804 bp of pEUCCR) labelled by random priming withdCTPα³² P.

The hybridization conditions used for the screening are as follows:

the pre-hybridization is carried out in 0.25% Marvel, 5× SSPE, 0.05% SDSat 58° C. The hybridization is carried out in the same buffer, in thepresence of 50 ng of a probe labelled with dCTα³² P, at 58° C. Thefilters are then washed with 2× SSC/0.1% SDS for 20 minutes at ambienttemperature, 2× SSC/0.1% SDS for 20 minutes at 58° C., 1× SSC/0.1% SDSfor 15 minutes at 58° C.

The plasmid pBluescript SK⁻ containing the cDNA clone (cloned in theEcoR1 site, using EcoR1 adaptors), was excised in vivo.

The cDNA is represented by SEQ ID NO 7, and the derived amino acidsequence of this cDNA is represented by SEQ ID NO 8.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 10                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1297 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 136..1140                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CGGCCGGGAC GACCCGTTCC TCTTCTTCCG GGTCACCGTC ACCATGTTAC AC - #AACATCTC         60                                                                          - CGGCTAAAAA AAAAAGGAAA AAAAGCGCAA CCTCCACCTC CTGAACCCCT CT - #CCCCCCTC        120                                                                          #GGT TCC GGC CAG ACC     171TC GAC GCC CTC CCC                                #Pro Val Asp Ala Leu Pro Gly Ser Gly Gln T - #hr                              #                10                                                           - GTC TGC GTC ACC GGC GCC GGC GGG TTC ATC GC - #C TCC TGG ATT GTC AAG          219                                                                          Val Cys Val Thr Gly Ala Gly Gly Phe Ile Al - #a Ser Trp Ile Val Lys           #         25                                                                  - CTT CTC CTC GAG CGA GGC TAC ACC GTG CGA GG - #A ACC GTC AGG AAC CCA          267                                                                          Leu Leu Leu Glu Arg Gly Tyr Thr Val Arg Gl - #y Thr Val Arg Asn Pro           #     40                                                                      - GAC GAC CCG AAG AAT GGT CAT CTG AGA GAT CT - #G GAA GGA GCC AGC GAG          315                                                                          Asp Asp Pro Lys Asn Gly His Leu Arg Asp Le - #u Glu Gly Ala Ser Glu           # 60                                                                          - AGG CTG ACG CTG TAC AAG GGT GAT CTG ATG GA - #C TAC GGG AGC TTG GAA          363                                                                          Arg Leu Thr Leu Tyr Lys Gly Asp Leu Met As - #p Tyr Gly Ser Leu Glu           #                 75                                                          - GAA GCC ATC AAG GGG TGC GAC GGC GTC GTC CA - #C ACC GCC TCT CCG GTC          411                                                                          Glu Ala Ile Lys Gly Cys Asp Gly Val Val Hi - #s Thr Ala Ser Pro Val           #             90                                                              - ACC GAC GAT CCT GAG CAA ATG GTG GAG CCA GC - #G GTG ATC GGG ACG AAA          459                                                                          Thr Asp Asp Pro Glu Gln Met Val Glu Pro Al - #a Val Ile Gly Thr Lys           #        105                                                                  - AAT GTG ATC GTC GCA GCG GCG GAG GCC AAG GT - #C CGG CGG GTT GTG TTC          507                                                                          Asn Val Ile Val Ala Ala Ala Glu Ala Lys Va - #l Arg Arg Val Val Phe           #   120                                                                       - ACC TCC TCC ATC GGT GCA GTC ACC ATG GAC CC - #C AAC CGG GCA GAC GTT          555                                                                          Thr Ser Ser Ile Gly Ala Val Thr Met Asp Pr - #o Asn Arg Ala Asp Val           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - GTG GTG GAC GAG TCT TGT TGG AGC GAC CTC GA - #A TTT TGC AAG AGC ACT          603                                                                          Val Val Asp Glu Ser Cys Trp Ser Asp Leu Gl - #u Phe Cys Lys Ser Thr           #               155                                                           - AAG AAC TGG TAT TGC TAC GGC AAG GCA GTG GC - #G GAG AAG GCC GCT TGG          651                                                                          Lys Asn Trp Tyr Cys Tyr Gly Lys Ala Val Al - #a Glu Lys Ala Ala Trp           #           170                                                               - CCA GAG GGC AAG GAG AGA GGG GTT GAC CTC GT - #G GTG ATT AAC CCT GTG          699                                                                          Pro Glu Gly Lys Glu Arg Gly Val Asp Leu Va - #l Val Ile Asn Pro Val           #       185                                                                   - CTC GTG CTT GGA CCG CTC CTT CAG TCG ACG AT - #C AAT GCG AGC ATC ATC          747                                                                          Leu Val Leu Gly Pro Leu Leu Gln Ser Thr Il - #e Asn Ala Ser Ile Ile           #   200                                                                       - CAC ATC CTC AAG TAC TTG ACT GGC TCA GCC AA - #G ACC TAC GCC AAC TCG          795                                                                          His Ile Leu Lys Tyr Leu Thr Gly Ser Ala Ly - #s Thr Tyr Ala Asn Ser           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - GTC CAG GCG TAC GTG CAC GTC AAG GAC GTC GC - #G CTT GCC CAC GTC CTT          843                                                                          Val Gln Ala Tyr Val His Val Lys Asp Val Al - #a Leu Ala His Val Leu           #               235                                                           - GTC TTG GAG ACC CCA TCC GCC TCA GGC CGC TA - #T TTG TGC GCC GAG AGC          891                                                                          Val Leu Glu Thr Pro Ser Ala Ser Gly Arg Ty - #r Leu Cys Ala Glu Ser           #           250                                                               - GTC CTC CAC CGT GGC GAT GTG GTG GAA ATC CT - #T GCC AAG TTC TTC CCT          939                                                                          Val Leu His Arg Gly Asp Val Val Glu Ile Le - #u Ala Lys Phe Phe Pro           #       265                                                                   - GAG TAT AAT GTA CCG ACC AAG TGC TCT GAT GA - #G GTG AAC CCA AGA GTA          987                                                                          Glu Tyr Asn Val Pro Thr Lys Cys Ser Asp Gl - #u Val Asn Pro Arg Val           #   280                                                                       - AAA CCA TAC AAG TTC TCC AAC CAG AAG CTG AG - #A GAC TTG GGG CTC GAG         1035                                                                          Lys Pro Tyr Lys Phe Ser Asn Gln Lys Leu Ar - #g Asp Leu Gly Leu Glu           285                 2 - #90                 2 - #95                 3 -       #00                                                                           - TTC ACC CCG GTG AAG CAG TGC CTG TAC GAA AC - #T GTC AAG AGC TTG CAG         1083                                                                          Phe Thr Pro Val Lys Gln Cys Leu Tyr Glu Th - #r Val Lys Ser Leu Gln           #               315                                                           - GAG AAA GGC CAC CTA CCA GTC CCC TCC CCG CC - #G GAA GAT TCG GTG CGT         1131                                                                          Glu Lys Gly His Leu Pro Val Pro Ser Pro Pr - #o Glu Asp Ser Val Arg           #           330                                                               - ATT CAG GGA TGATCTTAGA TCCATCACGG TGCGCATTTG AAATCCGGA - #G                 1180                                                                          Ile Gln Gly                                                                           335                                                                   - AAATGAGAGA AACATGTGGG AATTTGTTTG TACTTTTCTA AGTCAAACCT GG - #AGATACCA       1240                                                                          - ACCCTGAGTT CTGCATTGGA ATGGAAGTTG TCAATTGTTC CAAAAAAAAA AA - #AAAAA          1297                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 335 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Pro Val Asp Ala Leu Pro Gly Ser Gly Gl - #n Thr Val Cys Val Thr         #                 15                                                          - Gly Ala Gly Gly Phe Ile Ala Ser Trp Ile Va - #l Lys Leu Leu Leu Glu         #             30                                                              - Arg Gly Tyr Thr Val Arg Gly Thr Val Arg As - #n Pro Asp Asp Pro Lys         #         45                                                                  - Asn Gly His Leu Arg Asp Leu Glu Gly Ala Se - #r Glu Arg Leu Thr Leu         #     60                                                                      - Tyr Lys Gly Asp Leu Met Asp Tyr Gly Ser Le - #u Glu Glu Ala Ile Lys         # 80                                                                          - Gly Cys Asp Gly Val Val His Thr Ala Ser Pr - #o Val Thr Asp Asp Pro         #                 95                                                          - Glu Gln Met Val Glu Pro Ala Val Ile Gly Th - #r Lys Asn Val Ile Val         #           110                                                               - Ala Ala Ala Glu Ala Lys Val Arg Arg Val Va - #l Phe Thr Ser Ser Ile         #       125                                                                   - Gly Ala Val Thr Met Asp Pro Asn Arg Ala As - #p Val Val Val Asp Glu         #   140                                                                       - Ser Cys Trp Ser Asp Leu Glu Phe Cys Lys Se - #r Thr Lys Asn Trp Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Cys Tyr Gly Lys Ala Val Ala Glu Lys Ala Al - #a Trp Pro Glu Gly Lys         #               175                                                           - Glu Arg Gly Val Asp Leu Val Val Ile Asn Pr - #o Val Leu Val Leu Gly         #           190                                                               - Pro Leu Leu Gln Ser Thr Ile Asn Ala Ser Il - #e Ile His Ile Leu Lys         #       205                                                                   - Tyr Leu Thr Gly Ser Ala Lys Thr Tyr Ala As - #n Ser Val Gln Ala Tyr         #   220                                                                       - Val His Val Lys Asp Val Ala Leu Ala His Va - #l Leu Val Leu Glu Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ser Ala Ser Gly Arg Tyr Leu Cys Ala Gl - #u Ser Val Leu His Arg         #               255                                                           - Gly Asp Val Val Glu Ile Leu Ala Lys Phe Ph - #e Pro Glu Tyr Asn Val         #           270                                                               - Pro Thr Lys Cys Ser Asp Glu Val Asn Pro Ar - #g Val Lys Pro Tyr Lys         #       285                                                                   - Phe Ser Asn Gln Lys Leu Arg Asp Leu Gly Le - #u Glu Phe Thr Pro Val         #   300                                                                       - Lys Gln Cys Leu Tyr Glu Thr Val Lys Ser Le - #u Gln Glu Lys Gly His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Pro Val Pro Ser Pro Pro Glu Asp Ser Va - #l Arg Ile Gln Gly             #               335                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1376 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 99..1112                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GAAAACACAC CTCCTCTCTT CTTTGTCTCT GTCTGTTCTC CACTTTCCCA GT - #CACCAAAC         60                                                                          #GAT GCT        113ACAT TTATCTAAAT ATAACAAC ATG CCT GTT                       #      Met Pro Val Asp Ala                                                    #     5  1                                                                    - TCA TCA CTT TCA GGC CAA GGC CAA ACT ATC TG - #T GTC ACC GGG GGT GGT          161                                                                          Ser Ser Leu Ser Gly Gln Gly Gln Thr Ile Cy - #s Val Thr Gly Gly Gly           #                 20                                                          - GGT TTC ATT GCT TCT TGG ATG GTT AAA CTT CT - #T TTA GAT AAA GGT TAC          209                                                                          Gly Phe Ile Ala Ser Trp Met Val Lys Leu Le - #u Leu Asp Lys Gly Tyr           #             35                                                              - ACT GTT AGA GGA ACT GCG AGG AAC CCA GCT GA - #T CCC AAG AAT TCT CAT          257                                                                          Thr Val Arg Gly Thr Ala Arg Asn Pro Ala As - #p Pro Lys Asn Ser His           #         50                                                                  - TTG AGG GAG CTT GAA GGA GCT GAA GAA AGA TT - #A ACT TTA TGC AAA GCT          305                                                                          Leu Arg Glu Leu Glu Gly Ala Glu Glu Arg Le - #u Thr Leu Cys Lys Ala           #     65                                                                      - GAT CTT CTT GAT TAT GAG TCT CTT AAA GAG GG - #T ATT CAA GGG TGT GAT          353                                                                          Asp Leu Leu Asp Tyr Glu Ser Leu Lys Glu Gl - #y Ile Gln Gly Cys Asp           # 85                                                                          - GGT GTT TTC CAC ACT GCT TCT CCT GTC ACA GA - #T GAT CCG GAA GAA ATG          401                                                                          Gly Val Phe His Thr Ala Ser Pro Val Thr As - #p Asp Pro Glu Glu Met           #                100                                                          - GTG GAG CCA GCA GTG AAC GGG ACC AAA AAT GT - #G ATA ATT GCG GCG GCT          449                                                                          Val Glu Pro Ala Val Asn Gly Thr Lys Asn Va - #l Ile Ile Ala Ala Ala           #           115                                                               - GAG GCC AAA GTC CGA CGA GTG GTG TTC ACG TC - #A TCA ATT GGC GCT GTG          497                                                                          Glu Ala Lys Val Arg Arg Val Val Phe Thr Se - #r Ser Ile Gly Ala Val           #       130                                                                   - TAC ATG GAT CCC AAT AAG GGC CCA GAT GTT GT - #C ATT GAT GAG TCT TGC          545                                                                          Tyr Met Asp Pro Asn Lys Gly Pro Asp Val Va - #l Ile Asp Glu Ser Cys           #   145                                                                       - TGG AGT GAT CTT GAA TTC TGC AAG AAC ACC AA - #G AAT TGG TAT TGC TAT          593                                                                          Trp Ser Asp Leu Glu Phe Cys Lys Asn Thr Ly - #s Asn Trp Tyr Cys Tyr           150                 1 - #55                 1 - #60                 1 -       #65                                                                           - GGA AAG GCT GTG GCA GAA CAA GCT GCA TGG GA - #T ATG GCT AAG GAG AAA          641                                                                          Gly Lys Ala Val Ala Glu Gln Ala Ala Trp As - #p Met Ala Lys Glu Lys           #               180                                                           - GGG GTG GAC CTA GTG GTG GTT AAC CCA GTG CT - #G GTG CTT GGA CCA TTG          689                                                                          Gly Val Asp Leu Val Val Val Asn Pro Val Le - #u Val Leu Gly Pro Leu           #           195                                                               - TTG CAG CCC ACT GTC AAT GCT AGC ATC ACT CA - #C ATC CTC AAG TAC CTC          737                                                                          Leu Gln Pro Thr Val Asn Ala Ser Ile Thr Hi - #s Ile Leu Lys Tyr Leu           #       210                                                                   - ACC GGC TCA GCC AAG ACA TAT GCT AAC TCT GT - #T CAA GCT TAT GTG CAT          785                                                                          Thr Gly Ser Ala Lys Thr Tyr Ala Asn Ser Va - #l Gln Ala Tyr Val His           #   225                                                                       - GTT AGG GAT GTG GCA CTA GCC CAC ATT TTA GT - #C TTT GAG ACG CCT TCC          833                                                                          Val Arg Asp Val Ala Leu Ala His Ile Leu Va - #l Phe Glu Thr Pro Ser           230                 2 - #35                 2 - #40                 2 -       #45                                                                           - GCC TCC GGC CGT TAC CTT TGC TCT GAG AGC GT - #T CTC CAC CGT GGA GAG          881                                                                          Ala Ser Gly Arg Tyr Leu Cys Ser Glu Ser Va - #l Leu His Arg Gly Glu           #               260                                                           - GTG GTG GAA ATC CTT GCA AAG TTC TTC CCT GA - #G TAC CCC ATC CCT ACC          929                                                                          Val Val Glu Ile Leu Ala Lys Phe Phe Pro Gl - #u Tyr Pro Ile Pro Thr           #           275                                                               - AAG TGC TCA GAT GAG AAG AAC CCA AGA AAA CA - #A CCT TAC AAG TTC TCA          977                                                                          Lys Cys Ser Asp Glu Lys Asn Pro Arg Lys Gl - #n Pro Tyr Lys Phe Ser           #       290                                                                   - AAC CAG AAG CTA AGG GAT CTG GGT TTC GAA TT - #C ACC CCA GTA AAG CAG         1025                                                                          Asn Gln Lys Leu Arg Asp Leu Gly Phe Glu Ph - #e Thr Pro Val Lys Gln           #   305                                                                       - TGT CTG TAT GAA ACT GTT AAG AGT TTG CAG GA - #A AAG GGT CAC CTT CCA         1073                                                                          Cys Leu Tyr Glu Thr Val Lys Ser Leu Gln Gl - #u Lys Gly His Leu Pro           310                 3 - #15                 3 - #20                 3 -       #25                                                                           - ATC CCA AAA CAA GCT GCA GAA GAG TCT TTG AA - #A ATT CAA TAAGGCCTCT          1122                                                                          Ile Pro Lys Gln Ala Ala Glu Glu Ser Leu Ly - #s Ile Gln                       #               335                                                           - TGGAACTATT TATTAGGATT GTTCCATACC CCAAGTTTGG ATCGCAAATG CT - #AGGGAAAG       1182                                                                          - GAGCATATTA AAGAATGCCA ATGTGCAGGT GTTTTAGTAT TTTACATGAA GA - #ACTCTGAT       1242                                                                          - TATCCTTGTG CTTATAATAA TTTTTTTCAA GTGAGTGTCT TCAAATGTTC AA - #CTTGTATT       1302                                                                          - TGTGGTTGTC TAACTTTATC CAGTTTCAAT ATAAAAGAGG AACGATTCTA TG - #TCTTAAAA       1362                                                                          #   1376                                                                      - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 338 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Pro Val Asp Ala Ser Ser Leu Ser Gly Gl - #n Gly Gln Thr Ile Cys         #                 15                                                          - Val Thr Gly Gly Gly Gly Phe Ile Ala Ser Tr - #p Met Val Lys Leu Leu         #             30                                                              - Leu Asp Lys Gly Tyr Thr Val Arg Gly Thr Al - #a Arg Asn Pro Ala Asp         #         45                                                                  - Pro Lys Asn Ser His Leu Arg Glu Leu Glu Gl - #y Ala Glu Glu Arg Leu         #     60                                                                      - Thr Leu Cys Lys Ala Asp Leu Leu Asp Tyr Gl - #u Ser Leu Lys Glu Gly         # 80                                                                          - Ile Gln Gly Cys Asp Gly Val Phe His Thr Al - #a Ser Pro Val Thr Asp         #                 95                                                          - Asp Pro Glu Glu Met Val Glu Pro Ala Val As - #n Gly Thr Lys Asn Val         #           110                                                               - Ile Ile Ala Ala Ala Glu Ala Lys Val Arg Ar - #g Val Val Phe Thr Ser         #       125                                                                   - Ser Ile Gly Ala Val Tyr Met Asp Pro Asn Ly - #s Gly Pro Asp Val Val         #   140                                                                       - Ile Asp Glu Ser Cys Trp Ser Asp Leu Glu Ph - #e Cys Lys Asn Thr Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Trp Tyr Cys Tyr Gly Lys Ala Val Ala Gl - #u Gln Ala Ala Trp Asp         #               175                                                           - Met Ala Lys Glu Lys Gly Val Asp Leu Val Va - #l Val Asn Pro Val Leu         #           190                                                               - Val Leu Gly Pro Leu Leu Gln Pro Thr Val As - #n Ala Ser Ile Thr His         #       205                                                                   - Ile Leu Lys Tyr Leu Thr Gly Ser Ala Lys Th - #r Tyr Ala Asn Ser Val         #   220                                                                       - Gln Ala Tyr Val His Val Arg Asp Val Ala Le - #u Ala His Ile Leu Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Glu Thr Pro Ser Ala Ser Gly Arg Tyr Le - #u Cys Ser Glu Ser Val         #               255                                                           - Leu His Arg Gly Glu Val Val Glu Ile Leu Al - #a Lys Phe Phe Pro Glu         #           270                                                               - Tyr Pro Ile Pro Thr Lys Cys Ser Asp Glu Ly - #s Asn Pro Arg Lys Gln         #       285                                                                   - Pro Tyr Lys Phe Ser Asn Gln Lys Leu Arg As - #p Leu Gly Phe Glu Phe         #   300                                                                       - Thr Pro Val Lys Gln Cys Leu Tyr Glu Thr Va - #l Lys Ser Leu Gln Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Gly His Leu Pro Ile Pro Lys Gln Ala Al - #a Glu Glu Ser Leu Lys         #               335                                                           - Ile Gln                                                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1273 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 66..1091                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - TCGTAGCTCT TCCCTTTCAC CAACAAGCTA GTTTAGACAA GTACAGTGGT AC - #TGTAAGAG         60                                                                          #CAG CTG CCT GGC CAT        107 GCC GCG CCG                                   #Ala Pro Gln Leu Pro Gly Hisa Ala                                             #     10                                                                      - GGG CAG ACC GTG TGC GTC ACC GGC GCC GCG GG - #G TAC ATC GCG TCG GGG          155                                                                          Gly Gln Thr Val Cys Val Thr Gly Ala Ala Gl - #y Tyr Ile Ala Ser Gly           # 30                                                                          - CTC GTC AAG CTG CTC CTG GAG AGA GGC TAC AC - #C GTG AAG GGC ACA GTG          203                                                                          Leu Val Lys Leu Leu Leu Glu Arg Gly Tyr Th - #r Val Lys Gly Thr Val           #                 45                                                          - AGG AAC CCA GAT GAT CCC AAG AAC GCC CAC CT - #G AAG GCG CTG GAC GGC          251                                                                          Arg Asn Pro Asp Asp Pro Lys Asn Ala His Le - #u Lys Ala Leu Asp Gly           #             60                                                              - GCC ACC AAG AGG CTG ATC CTC TGC AAA GCC GA - #C CTC CTC GAC TAC GAC          299                                                                          Ala Thr Lys Arg Leu Ile Leu Cys Lys Ala As - #p Leu Leu Asp Tyr Asp           #         75                                                                  - GCC ATA TGC GCC GCC GTC GAG GGC TGC CAC GG - #C GTG TTC CAC ACC GCC          347                                                                          Ala Ile Cys Ala Ala Val Glu Gly Cys His Gl - #y Val Phe His Thr Ala           #     90                                                                      - TCT CCA GTC ACC GAT GAT CCT GAG CAG ATG GT - #G GAG CCG GCG GTG CGG          395                                                                          Ser Pro Val Thr Asp Asp Pro Glu Gln Met Va - #l Glu Pro Ala Val Arg           #110                                                                          - GGC ACG GAG TAC GTG ATC AAC GCG GCA GCG GA - #T GCG GGA ACG GTG CGC          443                                                                          Gly Thr Glu Tyr Val Ile Asn Ala Ala Ala As - #p Ala Gly Thr Val Arg           #               125                                                           - CGG GTG GTG TTC ACG TCG TCA ATC GGT GCC AT - #C ACC ATG GAC CCC AAC          491                                                                          Arg Val Val Phe Thr Ser Ser Ile Gly Ala Il - #e Thr Met Asp Pro Asn           #           140                                                               - CGC GGT CCT GAC GTA GTC GTC AAT GAG TCC TG - #C TGG AGC GAC CTC GAA          539                                                                          Arg Gly Pro Asp Val Val Val Asn Glu Ser Cy - #s Trp Ser Asp Leu Glu           #       155                                                                   - TTC TGC AAG AAA ACC AAG AAC TGG TAC TGC TA - #C GGC AAG GCC GTG GCG          587                                                                          Phe Cys Lys Lys Thr Lys Asn Trp Tyr Cys Ty - #r Gly Lys Ala Val Ala           #   170                                                                       - GAG CAG GCT GCG TGG GAG GCG GCC AGG AAG CG - #C GGC ATC GAC CTC GTC          635                                                                          Glu Gln Ala Ala Trp Glu Ala Ala Arg Lys Ar - #g Gly Ile Asp Leu Val           175                 1 - #80                 1 - #85                 1 -       #90                                                                           - GTC GTG AAC CCT GTG CTC GTG GTA GGG CCG CT - #G CTG CAA CCA ACG GTG          683                                                                          Val Val Asn Pro Val Leu Val Val Gly Pro Le - #u Leu Gln Pro Thr Val           #               205                                                           - AAC GCT AGC GCC GCA CAC ATC CTC AAG TAC CT - #C GAC GGC TCG GCC AAG          731                                                                          Asn Ala Ser Ala Ala His Ile Leu Lys Tyr Le - #u Asp Gly Ser Ala Lys           #           220                                                               - AAG TAC GCC AAC GCT GTG CAG TCA TAC GTA GA - #C GTG CGT GAC GTA GCC          779                                                                          Lys Tyr Ala Asn Ala Val Gln Ser Tyr Val As - #p Val Arg Asp Val Ala           #       235                                                                   - GGC GCG CAC ATC CGG GTG TTC GAG GCG CCT GA - #G GCG TCG GGC CGG TAC          827                                                                          Gly Ala His Ile Arg Val Phe Glu Ala Pro Gl - #u Ala Ser Gly Arg Tyr           #   250                                                                       - CTC TGC GCC GAG CGC GTG CTG CAC CGT GGG GA - #C GTT GTC CAA ATC CTC          875                                                                          Leu Cys Ala Glu Arg Val Leu His Arg Gly As - #p Val Val Gln Ile Leu           255                 2 - #60                 2 - #65                 2 -       #70                                                                           - AGC AAA CTC TTG CCT GAG TAC CCT GTG CCA AC - #A AGG TGC TCT GAT GAA          923                                                                          Ser Lys Leu Leu Pro Glu Tyr Pro Val Pro Th - #r Arg Cys Ser Asp Glu           #               285                                                           - GTG AAC CCA CGG AAG CAG CCT TAT AAG ATG TC - #C AAC CAG AAG CTG CAG          971                                                                          Val Asn Pro Arg Lys Gln Pro Tyr Lys Met Se - #r Asn Gln Lys Leu Gln           #           300                                                               - GAT CTT GGC CTC CAG TTC ACT CCT GTG AAC GA - #C TCT CTG TAT GAG ACC         1019                                                                          Asp Leu Gly Leu Gln Phe Thr Pro Val Asn As - #p Ser Leu Tyr Glu Thr           #       315                                                                   - GTG AAG AGC CTC CAG GAG AAG GGA CAT CTC CT - #A GTA CCA AGC AAA CCC         1067                                                                          Val Lys Ser Leu Gln Glu Lys Gly His Leu Le - #u Val Pro Ser Lys Pro           #   330                                                                       - GAG GGA TTA AAC GGT GTA ACG GCA TGATACTGCT AA - #AGAAGCAG CAGAGTTCAC        1121                                                                          Glu Gly Leu Asn Gly Val Thr Ala                                               335                 3 - #40                                                   - GTGCTCCTGT AACATGGTCA AACATGAGTT GTTTTTCTGT ATAAATTCTA TC - #CAGTATCG       1181                                                                          - TGTTATTTAA GTGAACTAAG AGAACAGAAT ATTGTATCAT CTTCGATGTC CA - #ATACCTGG       1241                                                                          #        1273      CACC TAAAAAAAAA AA                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 342 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Thr Val Val Asp Ala Ala Ala Pro Gln Le - #u Pro Gly His Gly Gln         #                 15                                                          - Thr Val Cys Val Thr Gly Ala Ala Gly Tyr Il - #e Ala Ser Gly Leu Val         #             30                                                              - Lys Leu Leu Leu Glu Arg Gly Tyr Thr Val Ly - #s Gly Thr Val Arg Asn         #         45                                                                  - Pro Asp Asp Pro Lys Asn Ala His Leu Lys Al - #a Leu Asp Gly Ala Thr         #     60                                                                      - Lys Arg Leu Ile Leu Cys Lys Ala Asp Leu Le - #u Asp Tyr Asp Ala Ile         # 80                                                                          - Cys Ala Ala Val Glu Gly Cys His Gly Val Ph - #e His Thr Ala Ser Pro         #                 95                                                          - Val Thr Asp Asp Pro Glu Gln Met Val Glu Pr - #o Ala Val Arg Gly Thr         #           110                                                               - Glu Tyr Val Ile Asn Ala Ala Ala Asp Ala Gl - #y Thr Val Arg Arg Val         #       125                                                                   - Val Phe Thr Ser Ser Ile Gly Ala Ile Thr Me - #t Asp Pro Asn Arg Gly         #   140                                                                       - Pro Asp Val Val Val Asn Glu Ser Cys Trp Se - #r Asp Leu Glu Phe Cys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Lys Thr Lys Asn Trp Tyr Cys Tyr Gly Ly - #s Ala Val Ala Glu Gln         #               175                                                           - Ala Ala Trp Glu Ala Ala Arg Lys Arg Gly Il - #e Asp Leu Val Val Val         #           190                                                               - Asn Pro Val Leu Val Val Gly Pro Leu Leu Gl - #n Pro Thr Val Asn Ala         #       205                                                                   - Ser Ala Ala His Ile Leu Lys Tyr Leu Asp Gl - #y Ser Ala Lys Lys Tyr         #   220                                                                       - Ala Asn Ala Val Gln Ser Tyr Val Asp Val Ar - #g Asp Val Ala Gly Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - His Ile Arg Val Phe Glu Ala Pro Glu Ala Se - #r Gly Arg Tyr Leu Cys         #               255                                                           - Ala Glu Arg Val Leu His Arg Gly Asp Val Va - #l Gln Ile Leu Ser Lys         #           270                                                               - Leu Leu Pro Glu Tyr Pro Val Pro Thr Arg Cy - #s Ser Asp Glu Val Asn         #       285                                                                   - Pro Arg Lys Gln Pro Tyr Lys Met Ser Asn Gl - #n Lys Leu Gln Asp Leu         #   300                                                                       - Gly Leu Gln Phe Thr Pro Val Asn Asp Ser Le - #u Tyr Glu Thr Val Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Leu Gln Glu Lys Gly His Leu Leu Val Pr - #o Ser Lys Pro Glu Gly         #               335                                                           - Leu Asn Gly Val Thr Ala                                                                 340                                                               - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1293 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 95..1108                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - CCGAGCCTAT TTCTTCCCTA TATCCACTCA TCCTTGTCTT ATATCATCAT CA - #TCATCATC         60                                                                          - TACCTAAACC TGAGCTCAAC AGAAAAGTAA TACC ATG CCG TCA G - #TT TCC GGC            112                                                                          #  Met Pro Ser Val Ser Gly                                                    # 5  1                                                                        - CAA ATC GTT TGT GTT ACT GGC GCC GGA GGT TT - #C ATC GCC TCT TGG CTC          160                                                                          Gln Ile Val Cys Val Thr Gly Ala Gly Gly Ph - #e Ile Ala Ser Trp Leu           #             20                                                              - GTT AAA ATT CTT CTG GAA AAA GGC TAC ACT GT - #T AGA GGA ACA GTA CGA          208                                                                          Val Lys Ile Leu Leu Glu Lys Gly Tyr Thr Va - #l Arg Gly Thr Val Arg           #         35                                                                  - AAT CCA GAT GAT CGA AAA AAT AGT CAT TTG AG - #G GAG CTT GAA CGA GCA          256                                                                          Asn Pro Asp Asp Arg Lys Asn Ser His Leu Ar - #g Glu Leu Glu Arg Ala           #     50                                                                      - AAA GAG ACA TTG ACT CTG TGC AGA GCT GAT CT - #T CTT GAT TTT CAG AGT          304                                                                          Lys Glu Thr Leu Thr Leu Cys Arg Ala Asp Le - #u Leu Asp Phe Gln Ser           # 70                                                                          - TTG CGA GAA GCA ATC AGC GGC TGT GAC GGA GT - #T TTC CAC ACA CGT TCT          352                                                                          Leu Arg Glu Ala Ile Ser Gly Cys Asp Gly Va - #l Phe His Thr Arg Ser           #                 85                                                          - CCT GTC ACT GAT GAT CCA GAA CAA ATG GTG GA - #G CCA GCA GTT ATT GGT          400                                                                          Pro Val Thr Asp Asp Pro Glu Gln Met Val Gl - #u Pro Ala Val Ile Gly           #            100                                                              - ACA AAG AAT GTG ATA ACG GCA GCA GCA GAG GC - #C AAG GTG CGA CGT GTG          448                                                                          Thr Lys Asn Val Ile Thr Ala Ala Ala Glu Al - #a Lys Val Arg Arg Val           #       115                                                                   - GTG TTC ACT TCG TCA ATT GGT GCT GTG TAT AT - #G GAC CCA AAC AGG GAC          496                                                                          Val Phe Thr Ser Ser Ile Gly Ala Val Tyr Me - #t Asp Pro Asn Arg Asp           #   130                                                                       - CCT GAT AAG GTT GTC GAC GAG ACT TGT TGG AG - #T GAT CCT GAC TTC TGC          544                                                                          Pro Asp Lys Val Val Asp Glu Thr Cys Trp Se - #r Asp Pro Asp Phe Cys           135                 1 - #40                 1 - #45                 1 -       #50                                                                           - AAA AAC ACC AAG AAT TGG TAT TGT TAT GGG AA - #G ATG GTG GCA GAA CAA          592                                                                          Lys Asn Thr Lys Asn Trp Tyr Cys Tyr Gly Ly - #s Met Val Ala Glu Gln           #               165                                                           - GCA GCA TGG GAC GAA GCA AGG GAG AAA GGA GT - #C GAT TTG GTG GCA ATC          640                                                                          Ala Ala Trp Asp Glu Ala Arg Glu Lys Gly Va - #l Asp Leu Val Ala Ile           #           180                                                               - AAC CCA GTG TTG GTG CTT GGA CCA CTG CTC CA - #A CAG AAT GTG AAT GCC          688                                                                          Asn Pro Val Leu Val Leu Gly Pro Leu Leu Gl - #n Gln Asn Val Asn Ala           #       195                                                                   - AGT GTT CTT CAC ATC CAC AAG TAC CTA ACT GG - #C TCT GCT AAA ACA TAT          736                                                                          Ser Val Leu His Ile His Lys Tyr Leu Thr Gl - #y Ser Ala Lys Thr Tyr           #   210                                                                       - ACG TCC AAT TCA CTT CAG GCA TAT GTT CAT GT - #T AGG GAT GTG GCT TTA          784                                                                          Thr Ser Asn Ser Leu Gln Ala Tyr Val His Va - #l Arg Asp Val Ala Leu           215                 2 - #20                 2 - #25                 2 -       #30                                                                           - CGT CAC ATA CTT GTG TAC GAG ACA CCT TCT GC - #A TCT GGC CGT TAT CTC          832                                                                          Arg His Ile Leu Val Tyr Glu Thr Pro Ser Al - #a Ser Gly Arg Tyr Leu           #               245                                                           - TGT GCC GAG AGT GTG CTG CAT CGC TGC GAT GT - #G GTT GAA ATT CTC GCC          880                                                                          Cys Ala Glu Ser Val Leu His Arg Cys Asp Va - #l Val Glu Ile Leu Ala           #           260                                                               - AAA TTC TTC CCG GAG TAT CCT ATC CCC ACC AA - #G TGT TCA GAT GTG ACG          928                                                                          Lys Phe Phe Pro Glu Tyr Pro Ile Pro Thr Ly - #s Cys Ser Asp Val Thr           #       275                                                                   - AAG CCA AGG GTA AAA CCG TAC AAA TTC TCA AA - #C CAA AAG CTA AAG GAT          976                                                                          Lys Pro Arg Val Lys Pro Tyr Lys Phe Ser As - #n Gln Lys Leu Lys Asp           #   290                                                                       - TTG GGT CTG GAG TTT ACA CCA GTA CAA TGC TT - #A TAT GAA ACG GTG AAG         1024                                                                          Leu Gly Leu Glu Phe Thr Pro Val Gln Cys Le - #u Tyr Glu Thr Val Lys           295                 3 - #00                 3 - #05                 3 -       #10                                                                           - AGT CTA CAA GAG AAA GGT CAC CTT CCA ATT CC - #T ACT CAA AAG GAT GAG         1072                                                                          Ser Leu Gln Glu Lys Gly His Leu Pro Ile Pr - #o Thr Gln Lys Asp Glu           #               325                                                           - ATT ATT CGA ATT CAG TCT GAG AAA TTC AGA AG - #C TCT TAGCATGTAT              1118                                                                          Ile Ile Arg Ile Gln Ser Glu Lys Phe Arg Se - #r Ser                           #           335                                                               - TGAGGAAAAG GGATCAATGG TTAAAGTTGA CCATGGCGTT GTCCCTTTAT GT - #ACCAAGAC       1178                                                                          - CAAATGCACC TAGAAATTTA CTTGTCTACT CTGTTGTACT TTTACTTGTC AT - #GGAAATGT       1238                                                                          - TTTTAGTGTT TTCATTGTTA TGAGATATAT TTTGGTGTAA AAAAAAAAAA AA - #AAA            1293                                                                          - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 338 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Pro Ser Val Ser Gly Gln Ile Val Cys Va - #l Thr Gly Ala Gly Gly         #                 15                                                          - Phe Ile Ala Ser Trp Leu Val Lys Ile Leu Le - #u Glu Lys Gly Tyr Thr         #             30                                                              - Val Arg Gly Thr Val Arg Asn Pro Asp Asp Ar - #g Lys Asn Ser His Leu         #         45                                                                  - Arg Glu Leu Glu Arg Ala Lys Glu Thr Leu Th - #r Leu Cys Arg Ala Asp         #     60                                                                      - Leu Leu Asp Phe Gln Ser Leu Arg Glu Ala Il - #e Ser Gly Cys Asp Gly         # 80                                                                          - Val Phe His Thr Arg Ser Pro Val Thr Asp As - #p Pro Glu Gln Met Val         #                 95                                                          - Glu Pro Ala Val Ile Gly Thr Lys Asn Val Il - #e Thr Ala Ala Ala Glu         #           110                                                               - Ala Lys Val Arg Arg Val Val Phe Thr Ser Se - #r Ile Gly Ala Val Tyr         #       125                                                                   - Met Asp Pro Asn Arg Asp Pro Asp Lys Val Va - #l Asp Glu Thr Cys Trp         #   140                                                                       - Ser Asp Pro Asp Phe Cys Lys Asn Thr Lys As - #n Trp Tyr Cys Tyr Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Met Val Ala Glu Gln Ala Ala Trp Asp Gl - #u Ala Arg Glu Lys Gly         #               175                                                           - Val Asp Leu Val Ala Ile Asn Pro Val Leu Va - #l Leu Gly Pro Leu Leu         #           190                                                               - Gln Gln Asn Val Asn Ala Ser Val Leu His Il - #e His Lys Tyr Leu Thr         #       205                                                                   - Gly Ser Ala Lys Thr Tyr Thr Ser Asn Ser Le - #u Gln Ala Tyr Val His         #   220                                                                       - Val Arg Asp Val Ala Leu Arg His Ile Leu Va - #l Tyr Glu Thr Pro Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Ser Gly Arg Tyr Leu Cys Ala Glu Ser Va - #l Leu His Arg Cys Asp         #               255                                                           - Val Val Glu Ile Leu Ala Lys Phe Phe Pro Gl - #u Tyr Pro Ile Pro Thr         #           270                                                               - Lys Cys Ser Asp Val Thr Lys Pro Arg Val Ly - #s Pro Tyr Lys Phe Ser         #       285                                                                   - Asn Gln Lys Leu Lys Asp Leu Gly Leu Glu Ph - #e Thr Pro Val Gln Cys         #   300                                                                       - Leu Tyr Glu Thr Val Lys Ser Leu Gln Glu Ly - #s Gly His Leu Pro Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Thr Gln Lys Asp Glu Ile Ile Arg Ile Gl - #n Ser Glu Lys Phe Arg         #               335                                                           - Ser Ser                                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1297 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 136..1140                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - CGGCCGGGAC GACCCGTTCC TCTTCTTCCG GGTCACCGTC ACCATGTTAC AC - #AACATCTC         60                                                                          - CGGCTAAAAA AAAAAGGAAA AAAAGCGCAA CCTCCACCTC CTGAACCCCT CT - #CCCCCCTC        120                                                                          #GGT TCC GGC CAG ACC     171TC GAC GCC CTC CCC                                #Pro Val Asp Ala Leu Pro Gly Ser Gly Gln T - #hr                              #                10                                                           - GTC TGC GTC ACC GGC GCC GGC GGG TTC ATC GC - #C TCC TGG ATT GTC AAG          219                                                                          Val Cys Val Thr Gly Ala Gly Gly Phe Ile Al - #a Ser Trp Ile Val Lys           #         25                                                                  - CTT CTC CTC GAG CGA GGC TAC ACC GTG CGA GG - #A ACC GTC AGG AAC CCA          267                                                                          Leu Leu Leu Glu Arg Gly Tyr Thr Val Arg Gl - #y Thr Val Arg Asn Pro           #     40                                                                      - GAC GAC CCG AAG AAT GGT CAT CTG AGA GAT CT - #G GAA GGA GCC AGC GAG          315                                                                          Asp Asp Pro Lys Asn Gly His Leu Arg Asp Le - #u Glu Gly Ala Ser Glu           # 60                                                                          - AGG CTG ACG CTG TAC AAG GGT GAT CTG ATG GA - #C GAC GGG AGC TTG GAA          363                                                                          Arg Leu Thr Leu Tyr Lys Gly Asp Leu Met As - #p Asp Gly Ser Leu Glu           #                 75                                                          - GAA GCC ATC AAG GGG TGC GAC GGC GTC GTC CA - #C ACC GCC TCT CCG GTC          411                                                                          Glu Ala Ile Lys Gly Cys Asp Gly Val Val Hi - #s Thr Ala Ser Pro Val           #             90                                                              - ACC GAC GAT CCT GAG CAA ATG GTG GAG CCA GC - #G GTG ATC GGG ACG AAA          459                                                                          Thr Asp Asp Pro Glu Gln Met Val Glu Pro Al - #a Val Ile Gly Thr Lys           #        105                                                                  - AAT GTG ATC GTC GCA GCG GCG GAG GCC AAG GT - #C CGG CGG GTT GTG TTC          507                                                                          Asn Val Ile Val Ala Ala Ala Glu Ala Lys Va - #l Arg Arg Val Val Phe           #   120                                                                       - ACC TCC TCC ATC GGT GCA GTC ACC ATG GAC CC - #C AAC CGG GCA GAC GTT          555                                                                          Thr Ser Ser Ile Gly Ala Val Thr Met Asp Pr - #o Asn Arg Ala Asp Val           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - GTG GTG GAC GAG TCT TGT TGG AGC GAC CTC GA - #A TTT TGC AAG AGC ACT          603                                                                          Val Val Asp Glu Ser Cys Trp Ser Asp Leu Gl - #u Phe Cys Lys Ser Thr           #               155                                                           - AAG AAC TGG TAT TGC TAC GGC AAG GCA GTG GC - #G GAG AAG GCC GCT TGG          651                                                                          Lys Asn Trp Tyr Cys Tyr Gly Lys Ala Val Al - #a Glu Lys Ala Ala Trp           #           170                                                               - CCA GAG GGC AAG GAG AGA GGG GTT GAC CTC GT - #G GTG ATT AAC CCT GTG          699                                                                          Pro Glu Gly Lys Glu Arg Gly Val Asp Leu Va - #l Val Ile Asn Pro Val           #       185                                                                   - CTC GTG CTT GGA CCG CTC CTT CAG TCG ACG AT - #C AAT GCG AGC ATC ATC          747                                                                          Leu Val Leu Gly Pro Leu Leu Gln Ser Thr Il - #e Asn Ala Ser Ile Ile           #   200                                                                       - CAC ATC CTC AAG TAC TTG ACT GGC TCA GCC AA - #G ACC TAC GCC AAC TCG          795                                                                          His Ile Leu Lys Tyr Leu Thr Gly Ser Ala Ly - #s Thr Tyr Ala Asn Ser           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - GTC CAG GCG TAC GTG CAC GTC AAG GAC GTC GC - #G CTT GCC CAC GTC CTT          843                                                                          Val Gln Ala Tyr Val His Val Lys Asp Val Al - #a Leu Ala His Val Leu           #               235                                                           - GTC TTG GAG ACC CCA TCC GCC TCA GGC CGC TA - #T TTG TGC GCC GAG AGC          891                                                                          Val Leu Glu Thr Pro Ser Ala Ser Gly Arg Ty - #r Leu Cys Ala Glu Ser           #           250                                                               - GTC CTC CAC CGT GGC GAT GTG GTG GAA ATC CT - #T GCC AAG TTC TTC CCT          939                                                                          Val Leu His Arg Gly Asp Val Val Glu Ile Le - #u Ala Lys Phe Phe Pro           #       265                                                                   - GAG TAT AAT GTA CCG ACC AAG TGC TCT GAT GA - #G GTG AAC CCA AGA GTA          987                                                                          Glu Tyr Asn Val Pro Thr Lys Cys Ser Asp Gl - #u Val Asn Pro Arg Val           #   280                                                                       - AAA CCA TAC AAG TTC TCC AAC CAG AAG CTG AG - #A GAC TTG GGG CTC GAG         1035                                                                          Lys Pro Tyr Lys Phe Ser Asn Gln Lys Leu Ar - #g Asp Leu Gly Leu Glu           285                 2 - #90                 2 - #95                 3 -       #00                                                                           - TTC ACC CCG GTG AAG CAG TGC CTG TAC GAA AC - #T GTC AAG AGC TTG CAG         1083                                                                          Phe Thr Pro Val Lys Gln Cys Leu Tyr Glu Th - #r Val Lys Ser Leu Gln           #               315                                                           - GAG AAA GGC CAC CTA CCA GTC CCC TCC CCG CC - #G GAA GAT TCG GTG CGT         1131                                                                          Glu Lys Gly His Leu Pro Val Pro Ser Pro Pr - #o Glu Asp Ser Val Arg           #           330                                                               - ATT CAG GGA TGATCTTAGA TCCATCACGG TGCGCATTTG TAATCCGGA - #G                 1180                                                                          Ile Gln Gly                                                                           335                                                                   - AAATGAGAGA AACATGTGGG AATTTGTTTG TACTTTTCTA AGTCAAACCT GG - #AGATACCA       1240                                                                          - ACCCTGAGTT CTGCATTGGA ATGGAAGTTG TCAATTGTTC CAAAAAAAAA AA - #AAAAA          1297                                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 335 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Pro Val Asp Ala Leu Pro Gly Ser Gly Gl - #n Thr Val Cys Val Thr         #                 15                                                          - Gly Ala Gly Gly Phe Ile Ala Ser Trp Ile Va - #l Lys Leu Leu Leu Glu         #             30                                                              - Arg Gly Tyr Thr Val Arg Gly Thr Val Arg As - #n Pro Asp Asp Pro Lys         #         45                                                                  - Asn Gly His Leu Arg Asp Leu Glu Gly Ala Se - #r Glu Arg Leu Thr Leu         #     60                                                                      - Tyr Lys Gly Asp Leu Met Asp Asp Gly Ser Le - #u Glu Glu Ala Ile Lys         # 80                                                                          - Gly Cys Asp Gly Val Val His Thr Ala Ser Pr - #o Val Thr Asp Asp Pro         #                 95                                                          - Glu Gln Met Val Glu Pro Ala Val Ile Gly Th - #r Lys Asn Val Ile Val         #           110                                                               - Ala Ala Ala Glu Ala Lys Val Arg Arg Val Va - #l Phe Thr Ser Ser Ile         #       125                                                                   - Gly Ala Val Thr Met Asp Pro Asn Arg Ala As - #p Val Val Val Asp Glu         #   140                                                                       - Ser Cys Trp Ser Asp Leu Glu Phe Cys Lys Se - #r Thr Lys Asn Trp Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Cys Tyr Gly Lys Ala Val Ala Glu Lys Ala Al - #a Trp Pro Glu Gly Lys         #               175                                                           - Glu Arg Gly Val Asp Leu Val Val Ile Asn Pr - #o Val Leu Val Leu Gly         #           190                                                               - Pro Leu Leu Gln Ser Thr Ile Asn Ala Ser Il - #e Ile His Ile Leu Lys         #       205                                                                   - Tyr Leu Thr Gly Ser Ala Lys Thr Tyr Ala As - #n Ser Val Gln Ala Tyr         #   220                                                                       - Val His Val Lys Asp Val Ala Leu Ala His Va - #l Leu Val Leu Glu Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Ser Ala Ser Gly Arg Tyr Leu Cys Ala Gl - #u Ser Val Leu His Arg         #               255                                                           - Gly Asp Val Val Glu Ile Leu Ala Lys Phe Ph - #e Pro Glu Tyr Asn Val         #           270                                                               - Pro Thr Lys Cys Ser Asp Glu Val Asn Pro Ar - #g Val Lys Pro Tyr Lys         #       285                                                                   - Phe Ser Asn Gln Lys Leu Arg Asp Leu Gly Le - #u Glu Phe Thr Pro Val         #   300                                                                       - Lys Gln Cys Leu Tyr Glu Thr Val Lys Ser Le - #u Gln Glu Lys Gly His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Pro Val Pro Ser Pro Pro Glu Asp Ser Va - #l Arg Ile Gln Gly             #               335                                                           __________________________________________________________________________

We claim:
 1. A method of producing transgenic plants within which thebiosynthesis of lignins is regulated either in the sense of an increase,or in the sense of a reduction of the lignin levels produced, relativeto the normal lignin levels produced in plants comprising:transformingplant cells with a recombinant nucleotide sequence comprising one ormore coding regions, wherein said coding regions are selected from thegroup consisting of: the nucleotide sequence represented by SEQ ID NO 1,coding for a mRNA, said mRNA coding for the CCR of eucalyptusrepresented by SEQ ID NO 2, the nucleotide sequence represented by SEQID NO 3, coding for a mRNA, said mRNA coding for the CCR of poplarrepresented by SEQ ID NO 4, the nucleotide sequence represented by SEQID NO 5, coding for a mRNA, said mRNA coding for the CCR of fescuerepresented by SEQ ID NO 6, the nucleotide sequence represented by SEQID NO 7, coding for a mRNA, said mRNA coding for the CCR of tobaccorepresented by SEQ ID NO 8, the nucleotide sequence represented by SEQID NO 9, coding for a mRNA, said mRNA coding for a protein representedby SEQ ID NO 10, derived from the eucalyptus CCR, and the nucleotidesequence complementary to that represented by SEQ ID NO 1, SEQ ID NO 3,SEQ ID NO 5, SEQ ID NO 7 or SEQ ID NO 9, said complementary sequencecoding for an anti sense mRNA capable of hybridizing with the mRNA codedby sequences SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQID NO 9, respectively.
 2. A DNA sequence comprising:the nucleotidesequence represented by SEQ ID NO 1, coding for a mRNA, said mRNA codingfor the CCR represented by SEQ ID NO 2, or the nucleotide sequencerepresented by SEQ ID NO 9, coding for a mRNA, said mRNA coding for aprotein represented by SEQ ID NO 10 derived from the eucalyptus CCR, orthe nucleotide sequence represented by SEQ ID NO 3, coding for a mRNA,said mRNA coding for the CCR represented by SEQ ID NO 4, or thenucleotide sequence represented by SEQ ID NO 5, coding for a mRNA, saidmRNA coding for the CCR represented by SEQ ID NO 6, or the nucleotidesequence represented by SEQ ID NO 7, coding for a mRNA, said mRNA codingfor the CCR represented by SEQ ID NO
 8. 3. A DNA sequence comprising:thenucleotide sequence complementary to that represented by SEQ ID NO 1,said complementary sequence coding for an anti sense mRNA capable ofhybridizing with a mRNA which codes for the CCR represented by SEQ ID NO2, or the nucleotide sequence complementary to that represented by SEQID NO 9, said complementary sequence coding for an anti sense mRNAcapable of hybridizing with a mRNA which codes for the CCR representedby SEQ ID NO 10, or the nucleotide sequence complementary to thatrepresented by SEQ ID NO 3, said complementary sequence coding for ananti sense mRNA capable of hybridizing with the mRNA coding for the CCRrepresented by SEQ ID NO 4, or the nucleotide sequence complementary tothat represented by SEQ ID NO 5, said complementary sequence coding foran anti sense mRNA capable of hybridizing with the mRNA coding for theCCR represented by SEQ ID NO 6, or the nucleotide sequence complementaryto that represented by SEQ ID NO 7, said complementary sequence codingfor an anti sense mRNA capable of hybridizing with the mRNA coding forthe CCR represented by SEQ ID NO
 8. 4. A mRNA selected from the groupconsisting of:the mRNA coded by the DNA sequence represented by SEQ IDNO 1, the mRNA coded by the DNA sequence represented by SEQ ID NO 3, themRNA coded by the DNA sequence represented by SEQ ID NO 5, the mRNAcoded by the DNA sequence represented by SEQ ID NO 7, and the mRNA codedby the DNA sequence represented by SEQ ID NO
 9. 5. An anti sense mRNAcomprising nucleotides complementary to a mRNA according to claim 4,said anti sense mRNA capable of hybridizing with said mRNA.
 6. Arecombinant CCR from eucalyptus, poplar, tall fescue or tobaccocomprising SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 8,respectively.
 7. A complex formed between an anti sense mRNA accordingto claim 5, and a CCR mRNA.
 8. A recombinant nucleotide sequencecomprising at least one DNA sequence according to claim 2, said sequenceinserted in a heterologous sequence.
 9. A recombinant nucleotidesequence, comprising at least one complementary DNA sequence accordingto claim 3, inserted in a heterologous sequence.
 10. A recombinantnucleotide sequence according to claim 8 comprising the necessaryelements to regulate the expression of the nucleotide sequence saidnecessary elements comprising a promoter or a terminator oftranscription and if appropriate, at least one DNA sequence coding forall or part of an enzyme other than CCR which is involved in a stage ofthe biosynthesis of lignins in plants, in particular the mRNA coding forcinnamyl alcohol dehydrogenase (CAD), or at least one sequence codingfor all or part of the anti sense mRNA capable of hybridizing with saidmRNA, in particular with the mRNA coding for CAD.
 11. A recombinantvector comprising a recombinant nucleotide sequence according to claim10, integrated in one of its sites of its genome which is non essentialfor its replication.
 12. A process for the regulation of thebiosynthesis of lignins in plants, either by reducing, or by increasingthe levels of lignin produced, relative to the normal levels of ligninsproduced in these plants, said process comprising a transformation stageof cells of these plants using a vector containing:a nucleotide sequencecoding for an mRNA, said mRNA coding for a CCR in plants, or anucleotide sequence complementary to the nucleotide sequence coding forsaid mRNA, said complementary sequence coding for an anti sense mRNAcapable of hybridizing with said mRNA.
 13. A process for the reductionof the biosynthesis of lignin in plants and therefore the reduction ofthe levels of lignins produced relative to the normal levels of ligninsproduced in plants, comprising the transformation of the genome of saidplants, by incorporating:at least one DNA sequence according to claim 3,and, if appropriate, at least one DNA sequence coding for all or part ofan anti sense mRNA capable of hybridizing with a mRNA coding for anenzyme other than CCR, which is implicated in a stage of thebiosynthesis of lignins in plants, in particular the mRNA coding forCAD.
 14. A process for the reduction of the biosynthesis of lignin inplants and therefore the reduction of the levels of lignins producedrelative to the normal levels of lignins produced in plants, comprisingthe transformation of the genome of said plants, by incorporating:atleast one DNA sequence according to claim 2, and, if appropriate, atleast one DNA sequence coding for all or part of an enzyme other thanCCR, which is implicated in a stage of the biosynthesis of lignins inplants, in particular a DNA sequence coding for all or part of CAD. 15.A process for increasing the biosynthesis of lignin in plants, andtherefore increasing the levels of lignins produced in plants relativeto the normal lignin levels produced in plants, comprising thetransformation of the genome of these plants, by incorporating:at leastone DNA sequence according to claim 2, and, if appropriate, at least oneDNA sequence coding for an enzyme other than CCR, which is implicated ina stage of the biosynthesis of lignins in plants, in particular a DNAsequence coding for CAD.
 16. A plant, plant fragment, cell, fruit, seed,or pollen, transformed by incorporation of at least one nucleotidesequence selected from the group consisting of:the nucleotide sequencerepresented by SEQ ID NO 1, coding for a mRNA, said mRNA coding for theCCR of eucalyptus represented by SEQ ID NO 2, the nucleotide sequencerepresented by SEQ ID NO 3, coding for a mRNA, said mRNA coding for theCCR of poplar represented by SEQ ID NO 4, the nucleotide sequencerepresented by SEQ ID NO 5, coding for a mRNA, said mRNA coding for theCCR of fescue represented by SEQ ID NO 6, the nucleotide sequencerepresented by SEQ ID NO 7, coding for a mRNA, said mRNA coding for theCCR of tobacco represented by SEQ ID NO 8, the nucleotide sequencerepresented by SEQ ID NO 9, coding for a mRNA, said mRNA coding for aprotein represented by SEQ ID NO 10 derived from the eucalyptus CCR, andthe nucleotide sequence complementary to that represented by SEQ ID NO1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 or SEQ ID NO 9, saidcomplementary sequence coding for an anti sense mRNA capable ofhybridizing with the mRNA coded by sequences SEQ ID NO 1, SEQ ID NO 3,SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9, respectively.
 17. Arecombinant polypeptide, comprising SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO6 or SEQ ID NO 8, obtained by transformation of plant cells byintegrating a recombinant nucleotide sequence.
 18. The process of claim12, wherein said nucleotide sequence is selected from the groupconsisting of:the nucleotide sequence represented by SEQ ID NO 1, codingfor a mRNA, said mRNA coding for the CCR of eucalyptus represented bySEQ ID NO 2, the nucleotide sequence represented by SEQ ID NO 3, codingfor a mRNA, said mRNA coding for the CCR of poplar represented by SEQ IDNO 4, the nucleotide sequence represented by SEQ ID NO 5, coding for amRNA, said mRNA coding for the CCR of fescue represented by SEQ ID NO 6,the nucleotide sequence represented by SEQ ID NO 7, coding for a mRNA,said mRNA coding for the CCR of tobacco represented by SEQ ID NO 8, thenucleotide sequence represented by SEQ ID NO 9, coding for a mRNA, saidmRNA coding for a protein represented by SEQ ID NO 10 derived from theeucalyptus CCR, and the nucleotide sequence complementary to thatrepresented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 or SEQID NO 9, said complementary sequence coding for an anti sense mRNAcapable of hybridizing with the mRNA coded by sequences SEQ ID NO 1, SEQID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9, respectively.
 19. Arecombinant nucleotide sequence according to claim 9 comprising thenecessary elements to regulate the expression of the nucleotide sequencesaid necessary elements comprising a promoter or a terminator oftranscription and if appropriate, at least one DNA sequence coding forall or part of an enzyme other than CCR which is involved in a stage ofthe biosynthesis of lignins in plants, in particular the mRNA coding forcinnamyl alcohol dehydrogenase (CAD), or at least one sequence codingfor all or part of the anti sense mRNA capable of hybridizing with saidmRNA, in particular with the mRNA coding for CAD.
 20. A recombinantvector comprising a recombinant nucleotide sequence according to claim19, integrated in one of its sites of its genome which is non essentialfor its replication.